Methods for determining fraction of fetal nucleic acids in maternal samples

ABSTRACT

The invention provides compositions and methods for determining the fraction of fetal nucleic acids in a maternal sample comprising a mixture of fetal and maternal nucleic acids. The fraction of fetal nucleic acids can be used in determining the presence or absence of fetal aneuploidy.

CROSS-REFERENCE

This Application claims priority to U.S. Provisional Application Ser.No. 61/296,358 entitled “Methods for Determining Fraction of FetalNucleic Acids in Maternal Samples”, filed on Jan. 19, 2010; U.S.Provisional Application Ser. No. 61/360,837 entitled “Methods forDetermining Fraction of Fetal Nucleic Acids in Maternal Samples”, filedon Jul. 1, 2010; U.S. Provisional Application Ser. No. 61/407,017entitled “Method for Determining Copy Number Variations”, filed on Oct.26, 2010; and U.S. Provisional Application Ser. No. 61/455,849 entitled“Simultaneous determination of Aneuploidy and Fetal Fraction”, filed onOct. 26, 2010; which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2011, isnamed 32770201.txt and is 238,572 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for detectingfetal nucleic acids in a maternal sample and determining the fraction ofcell-free fetal nucleic acid circulating in a maternal sample.

BACKGROUND OF THE INVENTION

Invasive prenatal tests are potentially harmful to the mother and to thefetus. Therefore, there is a need for the development of noninvasiveprenatal tests. Maternal blood can contain fetal cells (see e.g., U.S.Patent Application Publication No. 20080070792) and cell-free fetal DNA(see e.g., Huang et al. (2008), Methods in Molecular Biology,444:203-208). While circulating fetal cells present an attractive targetfor non invasive prenatal diagnostics, particularly for the diagnosis offetal sex and chromosomal abnormalities by simple karyotyping, thescarcity of intact fetal cells in the maternal circulation (around onecell per ml of maternal blood), low efficiency of enrichment (Bianchi etal., Am J Hum Genet 61:822-829 [1997]) and difficulties with chromosomalanalysis associated with abnormally dense nuclei in some cells(Babochkina et al., Hematological 90:740-745 [2005]), have favoredresearch on cell-free DNA.

The establishment of the concentrations of cell-free fetal DNA (cfDNA)in maternal plasma in healthy pregnant women has formed the platform onwhich fetal DNA abnormalities in pregnancy-associated disorders can bestudied. The finding of a gradual increase in fetal DNA concentration inmaternal serum as gestation progresses has been shown to precedecomplications associated with preterm labor. A five-fold increase infetal DNA concentration has also been found in the serum obtained fromwomen affected by preeclampsia. Other pregnancy-related disorders thathave been linked to an elevated concentration of cfDNA includehyperemesis gravidarum (severe morning sickness), invasive placentation(in which the placenta contacts the maternal bloodstream), intrauterinegrowth restriction, feto-maternal hemorrhage and polyhydramnios. (WrightC.F. and Burton H., Human Reproduction Update 15(1):139-151 [2009]).

Quantitative analysis of cell free DNA by real-time PCR strategies hasalso indicated that the concentrations of circulatory fetal DNA areincreased in pregnancies with fetal aneuploidies, most notably trisomy21 (Lo et al., Clin Chem 45:1747-1751 [1999]). However, the fraction offetal DNA in maternal cell-free plasma DNA is usually determined bycomparing the amount of fetal-specific locus (such as the SRY locus onchromosome Y in male pregnancies) to that of a locus on any autosomethat is common to both the mother and the fetus by using quantitativereal-time PCR (Dahllan et al., Lancet 369:474-481 [2007]; Li et al.,Clin Chem 1002-1011 [2004]; Fan et al., Proc Natl Acad Sci105:16266-16271 [2008]).

Thus, there is a need for additional methods that would enable thedetermination of the fraction of fetal nucleic acid in both male andfemale pregnancies.

The method of the invention fulfills the need in providing the means todetermine fetal fraction that is independent of the gender of the fetus.The method can be applied for determining simultaneously the presence orabsence of a chromosomal aneuploidy or other copy number variation, andmay be used in conjunction with nay known methods that are used todetermine aneuploidies in maternal sample.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal nucleic acids. The fraction of fetalnucleic acids can be used in determining the presence or absence offetal aneuploidy.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction. The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Inanother embodiment, the plurality of polymorphic nucleic acids can belocated on different autosomes other than chromosomes 13, 18 and 21. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The maternal sample is selectedfrom blood, plasma, serum, urine and saliva. Preferably, the maternalsample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. For example, the plurality of polymorphic nucleic acids canbe located on a plurality of different chromosomes other thanchromosomes 13, 18, 21, X or Y. The maternal sample is selected fromblood, plasma, serum, urine and saliva. Preferably, the maternal sampleis a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The massivelyparallel sequencing is sequencing-by-synthesis with reversible dyeterminators. Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). Theplurality of polymorphic nucleic acids are located on a plurality ofdifferent chromosomes. In one embodiment, the plurality of polymorphicnucleic acids can be located on chromosomes 1-22 For example, theplurality of polymorphic nucleic acids can be located on a plurality ofdifferent chromosomes other than chromosomes 13, 18, 21, X or Y. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). Theplurality of polymorphic nucleic acids are located on a plurality ofdifferent chromosomes. In one embodiment, the plurality of polymorphicnucleic acids can be located on chromosomes 1-22. For example, theplurality of polymorphic nucleic acids can be located on a plurality ofdifferent chromosomes other than chromosomes 13, 18, 21, X or Y. Themassively parallel sequencing is sequencing-by-synthesis with reversibledye terminators. Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphicnucleic acids are located on a plurality of different chromosomes. Inone embodiment, the plurality of polymorphic nucleic acids can belocated on chromosomes 1-22. For example, the plurality of polymorphicnucleic acids can be located on a plurality of different chromosomesother than chromosomes 13, 18, 21, X or Y. The maternal sample isselected from blood, plasma, serum, urine and saliva. Preferably, thematernal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction. The pluralityof polymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). Theplurality of polymorphic nucleic acids are located on a plurality ofdifferent chromosomes. In one embodiment, the plurality of polymorphicnucleic acids can be located on chromosomes 1-22. For example, theplurality of polymorphic nucleic acids can be located on a plurality ofdifferent chromosomes other than chromosomes 13, 18, 21, X or Y. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). Theplurality of polymorphic nucleic acids are located on a plurality ofdifferent chromosomes. In one embodiment, the plurality of polymorphicnucleic acids can be located on chromosomes 1-22. For example, theplurality of polymorphic nucleic acids can be located on a plurality ofdifferent chromosomes other than chromosomes 13, 18, 21, X or Y. Themassively parallel sequencing is sequencing-by-synthesis with reversibledye terminators. Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction. The pluralityof polymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The maternal sample is selected from blood, plasma, serum, urineand saliva. Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction. The pluralityof polymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The massively parallel sequencing is sequencing-by-synthesis withreversible dye terminators. Alternatively, the massively parallelsequencing is sequencing-by-ligation or single molecule sequencing. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The maternal sample is selected from blood, plasma, serum, urineand saliva. Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The plurality ofpolymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The maternal sample is selected from blood, plasma, serum, urineand saliva. Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The plurality ofpolymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The massively parallel sequencing is sequencing-by-synthesis withreversible dye terminators. Alternatively, the massively parallelsequencing is sequencing-by-ligation or single molecule sequencing. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The massivelyparallel sequencing is sequencing-by-synthesis with reversible dyeterminators. Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic nucleic acids are located ona plurality of different chromosomes. In one embodiment, the pluralityof polymorphic nucleic acids can be located on chromosomes 1-22. Forexample, the plurality of polymorphic nucleic acids can be located on aplurality of different chromosomes other than chromosomes 13, 18, 21, Xor Y. The maternal sample is selected from blood, plasma, serum, urineand saliva. Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The plurality of polymorphic nucleicacids are located on a plurality of different chromosomes. In oneembodiment, the plurality of polymorphic nucleic acids can be located onchromosomes 1-22. For example, the plurality of polymorphic nucleicacids can be located on a plurality of different chromosomes other thanchromosomes 13, 18, 21, X or Y. The maternal sample is selected fromblood, plasma, serum, urine and saliva. Preferably, the maternal sampleis a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The massively parallelsequencing is sequencing-by-synthesis with reversible dye terminators.Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The plurality of polymorphic targetnucleic acids comprises at least one single nucleotide polymorphism(SNP). Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The plurality of polymorphic targetnucleic acids comprises at least one single nucleotide polymorphism(SNP). Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). The massively parallelsequencing is sequencing-by-synthesis with reversible dye terminators.Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). In someembodiments, the at least one SNP is a single SNP selected from rs560681(SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10),rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14),rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18),rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261(SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ IDNOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 &32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36),rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696(SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ IDNOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 &50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), andrs530022 (SEQ ID NOS 55 & 56). In other embodiments, the at least oneSNP is a tandem SNP selected from tandem SNP pairs rs7277033-rs2110153(SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ ID NOS 314 & 315);rs368657-rs376635 (SEQ ID NOS 316 & 317); rs2822731-rs2822732 (SEQ IDNOS 318 & 319); rs1475881-rs7275487 (SEQ ID NOS 320 & 321);rs1735976-rs2827016 (SEQ ID NOS 322 & 323); rs447340-rs2824097 (SEQ IDNOS 324 & 325); rs418989-rs13047336 (SEQ ID NOS 326 & 327);rs987980-rs987981 (SEQ ID NOS 328 & 329); rs4143392-rs4143391 (SEQ IDNOS 330 & 331); rs1691324-rs13050434 (SEQ ID NOS 332 & 333);rs11909758-rs9980111 (SEQ ID NOS 334 & 335); rs2826842-rs232414 (SEQ IDNOS 336 & 337); rs1980969-rs1980970 (SEQ ID NOS 338 & 339);rs9978999-rs9979175 (SEQ ID NOS 340 & 341); rs1034346-rs12481852 (SEQ IDNOS 342 & 343); rs7509629-rs2828358 (SEQ ID NOS 344 & 345);rs4817013-rs7277036 (SEQ ID NOS 346 & 347); rs9981121-rs2829696 (SEQ IDNOS 348 & 349); rs455921-rs2898102 (SEQ ID NOS 350 & 351);rs2898102-rs458848 (SEQ ID NOS 352 & 353); rs961301-rs2830208 (SEQ IDNOS 354 & 355); rs2174536-rs458076 (SEQ ID NOS 356 & 357);rs11088023-rs11088024 (SEQ ID NOS 358 & 359); rs1011734-rs1011733 (SEQID NOS 360 & 361); rs2831244-rs9789838 (SEQ ID NOS 362 & 363);rs8132769-rs2831440 (SEQ ID NOS 364 & 365); rs8134080-rs2831524 (SEQ IDNOS 366 & 367); rs4817219-rs4817220 (SEQ ID NOS 368 & 369);rs2250911-rs2250997 (SEQ ID NOS 370 & 371); rs2831899-rs2831900 (SEQ IDNOS 372 & 373); rs2831902-rs2831903 (SEQ ID NOS 374 & 375);rs11088086-rs2251447 (SEQ ID NOS 376 & 377); rs2832040-rs11088088 (SEQID NOS 378 & 379); rs2832141-rs2246777 (SEQ ID NOS 380 & 381);rs2832959-rs9980934 (SEQ ID NOS 382 & 383); rs2833734-rs2833735 (SEQ IDNOS 384 & 385); rs933121-rs933122 (SEQ ID NOS 386 & 387);rs2834140-rs12626953 (SEQ ID NOS 388 & 389); rs2834485-rs3453 (SEQ IDNOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS 392 & 393);rs2776266-rs2835001 (SEQ ID NOS 394 & 395); rs1984014-rs1984015 (SEQ IDNOS 396 & 397); rs7281674-rs2835316 (SEQ ID NOS 398 & 399);rs13047304-rs13047322 (SEQ ID NOS 400 & 401); rs2835545-rs4816551 (SEQID NOS 402 & 403); rs2835735-rs2835736 (SEQ ID NOS 404 & 405);rs13047608-rs2835826 (SEQ ID NOS 406 & 407); rs2836550-rs2212596 (SEQ IDNOS 408 & 409); rs2836660-rs2836661 (SEQ ID NOS 410 & 411);rs465612-rs8131220 (SEQ ID NOS 412 & 413); rs9980072-rs8130031 (SEQ IDNOS 414 & 415); rs418359-rs2836926 (SEQ ID NOS 416 & 417);rs7278447-rs7278858 (SEQ ID NOS 418 & 419); rs385787-rs367001 (SEQ IDNOS 420 & 421); rs367001-rs386095 (SEQ ID NOS 422 & 423);rs2837296-rs2837297 (SEQ ID NOS 424 & 425); and rs2837381-rs4816672 (SEQID NOS 426 & 427). The at least one STR is selected from CSF1PO, FGA,TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D21S11, D2S1338, Penta D, Penta E, D22S1045, D20S1082, D20S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. Thematernal sample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction. The plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP). Alternatively, the plurality of polymorphic targetnucleic acids comprises at least one short tandem repeat (STR). In someembodiments, the at least one SNP is a single SNP selected from rs560681(SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10),rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14),rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18),rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261(SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ IDNOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 &32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36),rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696(SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ IDNOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 &50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), andrs530022 (SEQ ID NOS 55 & 56). In other embodiments, the at least oneSNP is a tandem SNP selected from tandem SNP pairs rs7277033-rs2110153(SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ ID NOS 314 & 315);rs368657-rs376635 (SEQ ID NOS 316 & 317); rs2822731-rs2822732 (SEQ IDNOS 318 & 319); rs1475881-rs7275487 (SEQ ID NOS 320 & 321);rs1735976-rs2827016 (SEQ ID NOS 322 & 323); rs447340-rs2824097 (SEQ IDNOS 324 & 325); rs418989-rs13047336 (SEQ ID NOS 326 & 327);rs987980-rs987981 (SEQ ID NOS 328 & 329); rs4143392-rs4143391 (SEQ IDNOS 330 & 331); rs1691324-rs13050434 (SEQ ID NOS 332 & 333);rs11909758-rs9980111 (SEQ ID NOS 334 & 335); rs2826842-rs232414 (SEQ IDNOS 336 & 337); rs1980969-rs1980970 (SEQ ID NOS 338 & 339);rs9978999-rs9979175 (SEQ ID NOS 340 & 341); rs1034346-rs12481852 (SEQ IDNOS 342 & 343); rs7509629-rs2828358 (SEQ ID NOS 344 & 345);rs4817013-rs7277036 (SEQ ID NOS 346 & 347); rs9981121-rs2829696 (SEQ IDNOS 348 & 349); rs455921-rs2898102 (SEQ ID NOS 350 & 351);rs2898102-rs458848 (SEQ ID NOS 352 & 353); rs961301-rs2830208 (SEQ IDNOS 354 & 355); rs2174536-rs458076 (SEQ ID NOS 356 & 357);rs11088023-rs11088024 (SEQ ID NOS 358 & 359); rs1011734-rs1011733 (SEQID NOS 360 & 361); rs2831244-rs9789838 (SEQ ID NOS 362 & 363);rs8132769-rs2831440 (SEQ ID NOS 364 & 365); rs8134080-rs2831524 (SEQ IDNOS 366 & 367); rs4817219-rs4817220 (SEQ ID NOS 368 & 369);rs2250911-rs2250997 (SEQ ID NOS 370 & 371); rs2831899-rs2831900 (SEQ IDNOS 372 & 373); rs2831902-rs2831903 (SEQ ID NOS 374 & 375);rs11088086-rs2251447 (SEQ ID NOS 376 & 377); rs2832040-rs11088088 (SEQID NOS 378 & 379); rs2832141-rs2246777 (SEQ ID NOS 380 & 381);rs2832959-rs9980934 (SEQ ID NOS 382 & 383); rs2833734-rs2833735 (SEQ IDNOS 384 & 385); rs933121-rs933122 (SEQ ID NOS 386 & 387);rs2834140-rs12626953 (SEQ ID NOS 388 & 389); rs2834485-rs3453 (SEQ IDNOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS 392 & 393);rs2776266-rs2835001 (SEQ ID NOS 394 & 395); rs1984014-rs1984015 (SEQ IDNOS 396 & 397); rs7281674-rs2835316 (SEQ ID NOS 398 & 399);rs13047304-rs13047322 (SEQ ID NOS 400 & 401); rs2835545-rs4816551 (SEQID NOS 402 & 403); rs2835735-rs2835736 (SEQ ID NOS 404 & 405);rs13047608-rs2835826 (SEQ ID NOS 406 & 407); rs2836550-rs2212596 (SEQ IDNOS 408 & 409); rs2836660-rs2836661 (SEQ ID NOS 410 & 411);rs465612-rs8131220 (SEQ ID NOS 412 & 413); rs9980072-rs8130031 (SEQ IDNOS 414 & 415); rs418359-rs2836926 (SEQ ID NOS 416 & 417);rs7278447-rs7278858 (SEQ ID NOS 418 & 419); rs385787-rs367001 (SEQ IDNOS 420 & 421); rs367001-rs386095 (SEQ ID NOS 422 & 423);rs2837296-rs2837297 (SEQ ID NOS 424 & 425); and rs2837381-rs4816672 (SEQID NOS 426 & 427). The at least one STR is selected from CSF1PO, FGA,TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D21S11, D2S1338, Penta D, Penta E, D22S1045, D20S1082, D20S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. Themassively parallel sequencing is sequencing-by-synthesis with reversibledye terminators. Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction. The pluralityof polymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). In some embodiments, the at least one SNP is a single SNPselected from rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56). In other embodiments, the atleast one SNP is a tandem SNP selected from tandem SNP pairsrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427). The at least one STR isselected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113. The maternal sample is selected fromblood, plasma, serum, urine and saliva. Preferably, the maternal sampleis a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction. The pluralityof polymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). In some embodiments, the at least one SNP is a single SNPselected from rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56). In other embodiments, the atleast one SNP is a tandem SNP selected from tandem SNP pairsrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427). The at least one STR isselected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113. The massively parallel sequencing issequencing-by-synthesis with reversible dye terminators. Alternatively,the massively parallel sequencing is sequencing-by-ligation or singlemolecule sequencing. The maternal sample is selected from blood, plasma,serum, urine and saliva. Preferably, the maternal sample is a plasmasample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The maternal sampleis selected from blood, plasma, serum, urine and saliva. Preferably, thematernal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The plurality ofpolymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). In some embodiments, the at least one SNP is a single SNPselected from rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56). In other embodiments, the atleast one SNP is a tandem SNP selected from tandem SNP pairsrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427). The at least one STR isselected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113. The maternal sample is selected fromblood, plasma, serum, urine and saliva. Preferably, the maternal sampleis a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises providing aplurality of sequence tags; and (c) based on the sequencing, determiningthe fraction, wherein determining the fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising of at least one polymorphic nucleic acid. The plurality ofpolymorphic target nucleic acids comprises at least one singlenucleotide polymorphism (SNP). Alternatively, the plurality ofpolymorphic target nucleic acids comprises at least one short tandemrepeat (STR). In some embodiments, the at least one SNP is a single SNPselected from rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56). In other embodiments, the atleast one SNP is a tandem SNP selected from tandem SNP pairsrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427). The at least one STR isselected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113. The massively parallel sequencing issequencing-by-synthesis with reversible dye terminators. Alternatively,the massively parallel sequencing is sequencing-by-ligation or singlemolecule sequencing. The maternal sample is selected from blood, plasma,serum, urine and saliva. Preferably, the maternal sample is a plasmasample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The maternal sample isselected from blood, plasma, serum, urine and saliva. Preferably, thematernal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA, wherein the method comprisesthe steps of: (a) amplifying a plurality of polymorphic target nucleicacids in the mixture of fetal and maternal genomic DNA; (b) performingmassively parallel sequencing of at least a portion of the amplifiedproduct obtained in (a), wherein sequencing comprises an amplificationand provides a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The massively parallelsequencing is sequencing-by-synthesis with reversible dye terminators.Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The maternal sample is selected from blood, plasma, serum,urine and saliva. Preferably, the maternal sample is a plasma sample.

In one embodiment, the invention provides a method for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The maternal sample is selected fromblood, plasma, serum, urine and saliva. Preferably, the maternal sampleis a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The maternal sample isselected from blood, plasma, serum, urine and saliva. Preferably, thematernal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises providing a plurality of sequence tags; and (c) based on thesequencing, determining the fraction, wherein determining the fractioncomprises determining the number of fetal and maternal sequence tagsmapped to a reference genome comprising of at least one polymorphicnucleic acid. The plurality of polymorphic target nucleic acidscomprises at least one single nucleotide polymorphism (SNP).Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The massively parallelsequencing is sequencing-by-synthesis with reversible dye terminators.Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The plurality of polymorphic targetnucleic acids comprises at least one single nucleotide polymorphism(SNP). Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The maternal sample isselected from blood, plasma, serum, urine and saliva. Preferably, thematernal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal genomic DNA e.g. cell-free DNA, whereinthe method comprises the steps of: (a) amplifying a plurality ofpolymorphic target nucleic acids in the mixture of fetal and maternalgenomic DNA; (b) performing massively parallel sequencing of at least aportion of the amplified product obtained in (a), wherein sequencingcomprises an amplification and provides a plurality of sequence tags;and (c) based on the sequencing, determining the fraction, whereindetermining the fraction comprises determining the number of fetal andmaternal sequence tags mapped to a reference genome comprising of atleast one polymorphic nucleic acid. The plurality of polymorphic targetnucleic acids comprises at least one single nucleotide polymorphism(SNP). Alternatively, the plurality of polymorphic target nucleic acidscomprises at least one short tandem repeat (STR). In some embodiments,the at least one SNP is a single SNP selected from rs560681 (SEQ ID NOS1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56). In other embodiments, the at least one SNP is a tandem SNP selectedfrom tandem SNP pairs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427). The atleast one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. The massively parallelsequencing is sequencing-by-synthesis with reversible dye terminators.Alternatively, the massively parallel sequencing issequencing-by-ligation or single molecule sequencing. The maternalsample is selected from blood, plasma, serum, urine and saliva.Preferably, the maternal sample is a plasma sample.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The method is afetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises preamplifying the mixture of fetal and maternalnucleic acids. The method is a fetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises resolving the size of the STRs using capillaryelectrophoresis. The method is a fetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises preamplifying the mixture of fetal and maternalnucleic acids, and resolving the size of the STRs using capillaryelectrophoresis. The method is a fetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes. For example, the plurality of polymorphic nucleic acids arelocated on a plurality of different chromosomes other than chromosomes13, 18, 21, X or Y. The at least one STR is selected from CSF1PO, FGA,TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.Optionally, the at least one STR can be the panel of STRs comprisingCSF1PO, D13S317, D16 S539, D18S51, D21S11, D2S1338, D7S820 and FGA. Themethod is a fetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises preamplifying the mixture of fetal and maternalnucleic acids. The at least one STR is selected from CSF1PO, FGA, TH01,vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.Optionally, the at least one STR can be the panel of STRs comprisingCSF1PO, D13S317, D16S539, D18S51, D21S11, D2S1338, D7S820 and FGA. Themethod is a fetal gender-independent method.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises resolving the size of the STRs using capillaryelectrophoresis. The at least one STR is selected from CSF1PO, FGA,TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.Optionally, the at least one STR can be the panel of STRs comprisingCSF1PO, D13S317, D16S539, D18S51, D21S11, D2S1338, D7S820 and FGA.

In another embodiment, the invention provides a method for determiningthe fraction of fetal nucleic acids in a maternal plasma samplecomprising a mixture of fetal and maternal genomic DNA, wherein themethod comprises the steps of: (a) amplifying a plurality of polymorphicnucleic acids in said mixture of fetal and maternal nucleic acids,wherein each of said at least one polymorphic nucleic acid comprises anSTR; (b) determining the amount of fetal and maternal STR alleles atleast one polymorphic nucleic acid; and (c) determining said fractionusing said amount of fetal and maternal STR alleles. The plurality ofpolymorphic nucleic acids are located on a plurality of differentchromosomes. In one embodiment, the plurality of polymorphic nucleicacids can be located on chromosomes 1-22. For example, the plurality ofpolymorphic nucleic acids can be located on a plurality of differentchromosomes other than chromosomes 13, 18, 21, X or Y. The methodfurther comprises preamplifying the mixture of fetal and maternalnucleic acids, and resolving the size of the STRs using capillaryelectrophoresis. The at least one STR is selected from CSF1PO, FGA,TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.Optionally, the at least one STR can be the panel of STRs comprisingCSF1PO, D13S317, D16 S539, D18S51, D21S11, D2S1338, D7S820 and FGA.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The set of primers does not amplify a sequence on the Ychromosome.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The polymorphic nucleic acid comprises an STR, a SNP and/or atandem SNP.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The polymorphic nucleic acid comprises an STR selected fromCSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482,D18S853, D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435,D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408,D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, andD1GATA113; and/or a SNP selected from rs560681 (SEQ ID NOS 1 & 2),rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883(SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ IDNOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 &16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20),rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), rs2567608(SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171 (SEQ IDNOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ ID NOS 33 &34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 & 38),rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42), rs508485(SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254 (SEQ IDNOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS 51 &52), rs3739005 (SEQ ID NOS 53 & 54), rs530022 (SEQ ID NOS 55 & 56);and/or a tandem SNP rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959 rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample bymassively parallel sequencing the fetal and maternal cfDNA in thematernal sample, e.g. a plasma sample, wherein the composition comprisesat least one set of primers for amplifying at least one polymorphicnucleic acid in said mixture.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The polymorphic nucleic acid comprises an STR, a SNP and/or atandem SNP.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample bymassively parallel sequencing the fetal and maternal cfDNA in thematernal sample, e.g. a plasma sample, wherein the composition comprisesat least one set of primers for amplifying at least one polymorphicnucleic acid in said mixture. The polymorphic nucleic acid comprises anSTR selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820,D8S1179, D13S317, D16 S539, D18S51, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113; a SNP selected from rs560681 (SEQ IDNOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56); and/or a tandem SNP rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The set of primers does not amplify a sequence on the Ychromosome.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in themixture. The set of primers does not amplify a sequence on the Ychromosome. The polymorphic nucleic acid comprises an STR, a SNP and/ora tandem SNP.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The polymorphic nucleic acid comprises an STR selected fromCSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482,D18S853, D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435,D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408,D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, andD1GATA113; a SNP selected from rs560681 (SEQ ID NOS 1 & 2), rs1109037(SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12),rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16),rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20),rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), rs2567608(SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171 (SEQ IDNOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ ID NOS 33 &34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 & 38),rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42), rs508485(SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254 (SEQ IDNOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS 51 &52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 & 56);and/or a tandem SNP rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample bymassively parallel sequencing the fetal and maternal cIDNA in thematernal sample, e.g. a plasma sample, wherein the composition comprisesat least one set of primers for amplifying at least one polymorphicnucleic acid in said mixture.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample, e.g. aplasma sample, wherein the composition comprises at least one set ofprimers for amplifying at least one polymorphic nucleic acid in saidmixture. The set of primers does not amplify a sequence on the Ychromosome. The polymorphic nucleic acid comprises an STR, a SNP and/ora tandem SNP.

In another embodiment, the invention provides a composition fordetermining the fraction of fetal cfDNA in a maternal sample bymassively parallel sequencing the fetal and maternal cIDNA in thematernal sample, e.g. a plasma sample, wherein the composition comprisesat least one set of primers for amplifying at least one polymorphicnucleic acid in said mixture. The polymorphic nucleic acid comprises anSTR selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820,D8S1179, D13S317, D16 S539, D18S51, D2S1338, Penta D, Penta E, D22S1045,D20 S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113; a SNP selected from rs560681 (SEQ IDNOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6),rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158(SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ IDNOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 &20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24),rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171(SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ IDNOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 &38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42),rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254(SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 &56); and/or a tandem SNP rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959 rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).

In another embodiment, the invention provides a kit that comprises thecomposition of the invention as described above and in the following.

INCORPORATION BY REFERENCE

All patents, patent applications, and other publications, including allsequences disclosed within these references, referred to herein areexpressly incorporated by reference, to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference. Alldocuments cited are, in relevant part, incorporated herein by reference.However, the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flowchart of a method 100 for determining the fetal fractionin a maternal test sample comprising a mixture of fetal and maternalnucleic acids using massively parallel sequencing methods or sizeseparation of polymorphic nucleic acid sequences.

FIG. 2 is a bar diagram showing the identification of fetal and maternalpolymorphic sequences (SNPs) used to determine fetal fraction in a testsample. The total number of sequence reads (Y-axis) mapped to the SNPsequences identified by rs numbers (X-axis), and the relative level offetal nucleic acids (*) are shown.

FIG. 3 is a flowchart outlining alternative embodiments of the methodfor determining fetal fraction by massively parallel sequencing shown inFIG. 1.

FIG. 4 illustrates STR markers used in the AmpFλSTR® Identifiler® PCRAmplification Kit.

FIG. 5 illustrates STR markers used in the AmpFλSTR® MiniFiler® PCRAmplification Kit.

FIG. 6 illustrates the correlation of fetal fraction determined bymassively parallel sequencing size separation of polymorphic sequencescomprising SNPs and STRs.

FIG. 7 illustrates an embodiment of use of fetal fraction fordetermining cutoff thresholds for aneuploidy detection.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for determining thefraction of fetal nucleic acids in a maternal sample comprising amixture of fetal and maternal nucleic acids. The fraction of fetalnucleic acids can be used in determining the presence or absence offetal aneuploidy.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous standard texts and reference works. All patents,patent applications, articles and publications mentioned herein arehereby expressly incorporated herein by reference in their entirety.

Numeric ranges are inclusive of the numbers defining the range. It isintended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to theSpecification as a whole. Accordingly, as indicated above, the termsdefined immediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the present invention, some preferred methods andmaterials are described. Accordingly, the terms defined immediatelybelow are more fully described by reference to the Specification as awhole. It is to be understood that this invention is not limited to theparticular methodology, protocols, and reagents described, as these mayvary, depending upon the context they are used by those of skill in theart.

DEFINITIONS

As used herein, the singular terms “a”, “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation and amino acid sequences are written left to right in aminoto carboxy orientation, respectively.

As used herein, the singular terms “a”, “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation and amino acid sequences are written left to right in aminoto carboxy orientation, respectively.

The term “portion” when used in reference to the amount of sequenceinformation of fetal and maternal nucleic acid molecules in a biologicalsample herein refers to the amount of sequence information of fetal andmaternal nucleic acid molecules in a biological sample that in sumamount to less than the sequence information of <1 human genome.

The terms “polynucleotide”, “nucleic acid” and “nucleic acid molecules”are used interchangeably and refer to a covalently linked sequence ofnucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides forDNA) in which the 3′ position of the pentose of one nucleotide is joinedby a phosphodiester group to the 5′ position of the pentose of the next,include sequences of any form of nucleic acid, including, but notlimited to RNA, DNA and cfDNA molecules. The term “polynucleotide”includes, without limitation, single- and double-strandedpolynucleotide.

The term “copy number variation” herein refers to variation in thenumber of copies of a nucleic acid sequence that is 1 kb or largerpresent in a test sample in comparison with the copy number of thenucleic acid sequence present in a qualified sample. A “copy numbervariant” refers to the 1 kb or larger sequence of nucleic acid in whichcopy-number differences are found by comparison of a sequence ofinterest in test sample with that present in a qualified sample. Copynumber variants/variations include deletions, including microdeletions,insertions, including microinsertions, duplications, multiplications,inversions, translocations and complex multi-site variants. CNVencompass chromosomal aneuploidies and partial aneuplodies.

As used herein, the term “fetal fraction” is used interchangeably with“fraction of fetal nucleic acid”, which refers to the fraction of fetalnucleic acid in a sample comprising fetal and maternal nucleic acidSimilarly, the term “minor fraction” or “minor component” herein refersto the lesser fraction of the total genetic material that is present ina sample containing genetic material derived from separate sources e.g.individuals.

As used herein the term “allele” refers to one specific form of agenetic sequence (such as a gene) within a cell, a sample, an individualor within a population, the specific form differing from other forms ofthe same gene in the sequence of at least one, and frequently more thanone, variant sites within the sequence of the gene. The sequences atthese variant sites that differ between different alleles are termed“variants”, “polymorphisms”, or “mutations”. In general, polymorphism isused to refer to variants that have a frequency of at least 1% in apopulation, while the term mutation is generally used for variants thatoccur at a frequency of less than 1% in a population. In diploidorganisms such as humans, at each autosomal specific chromosomallocation or “locus” an individual possesses two alleles, a firstinherited from one parent and a second inherited from the other parent,for example one from the mother and one from the father. An individualis “heterozygous” at a locus if it has two different alleles at thelocus. An individual is “homozygous” at a locus if it has two identicalalleles at that locus.

The term “enrich” herein refers to the process of amplifying polymorphictarget nucleic acids contained in a portion of a maternal sample, andcombining the amplified product with the remainder of the maternalsample from which the portion was removed.

As used herein, the term “genotyping” refers to the determination of thegenetic information an individual carries at one or more positions inthe genome. For example, genotyping may comprise the determination ofwhich allele or alleles an individual carries for a single SNP or thedetermination of which allele or alleles an individual carries for aplurality of SNPs. For example, a particular nucleotide in a genome maybe a T in some individuals and a C in other individuals. Thoseindividuals who have a T at the position have the T allele and those whohave a C have the C allele. In a diploid organism the individual willhave two copies of the sequence containing the polymorphic position sothe individual may have a T allele and a C allele or alternatively twocopies of the T allele or two copies of the C allele. Those individualswho have two copies of the C allele are homozygous for the C allele,those individuals who have two copies of the T allele are homozygous forthe T allele, and those individuals who have one copy of each allele areheterozygous. The alleles are often referred to as the A allele, oftenthe major allele, and the B allele, often the minor allele. Thegenotypes may be AA (homozygous A), BB (homozygous B) or AB(heterozygous). Genotyping methods generally provide for identificationof the sample as AA, BB or AB.

As used herein the term “chromosome” refers to the heredity-bearing genecarrier of a living cell which is derived from chromatin and whichcomprises DNA and protein components (especially histones). Theconventional internationally recognized individual human genomechromosome numbering system is employed herein. The size of anindividual chromosome can vary from one type to another with a givenmulti-chromosomal genome and from one genome to another. In the case ofthe human genome, the entire DNA mass of a given chromosome is usuallygreater than about 100,000,000 bp. For example, the size of the entirehuman genome is about 3.times.10.sup.9 bp. The largest chromosome,chromosome no. 1, contains about 2.4.times.10.sup.8 by while thesmallest chromosome, chromosome no. 22, contains about5.3.times.10.sup.7 bp.

The term “aneuploidy” herein refers to the occurrence of one or moreextra or missing chromosomes.

As used herein the term “chromosomal region” is a portion of achromosome. The actual physical size or extent of any individualchromosomal region can vary greatly. The term “region” is notnecessarily definitive of a particular one or more genes because aregion need not take into specific account the particular codingsegments (exons) of an individual gene.

As used herein the term “genetic marker” refers to a sequence of DNAthat has a specific location on a chromosome that can be measured in alaboratory. The term “genetic marker” can also be used to refer to,e.g., a cDNA and/or an mRNA encoded by a genomic sequence, as well as tothat genomic sequence. To be useful, a marker needs to have two or morealleles or variants. Markers can be either direct, that is, locatedwithin the gene or locus of interest (i.e., candidate gene), orindirect, that is closely linked with the gene or locus of interest(presumably due to a location which is proximate to, but not inside thegene or locus of interest). Moreover, markers can also include sequenceswhich either do or do not modify the amino acid sequence of a gene.

As used herein, the term “maternal sample” refers to a biological sampleobtained from a pregnant subject, and comprises a mixture of fetal andmaternal nucleic acids. A “pregnant subject” is not limited to a humanbeing, but may also include other organisms including but not limited tomammals, plants, bacteria or cells derived from any of the above.

The term “whole genome amplification” or “WGA” as used herein generallyrefers to a method for amplification of a limited DNA sample in anon-specific manner, in order to generate a new sample that isindistinguishable from the original but with a higher DNA concentration.The ideal whole genome amplification technique would amplify a sample upto a microgram level while maintaining the original sequencerepresentation. The DNA of the sample may include an entire genome or aportion thereof. Degenerate oligonucleotide-primed PCR (DOP), primerextension PCR technique (PEP) including modified improved primerextension Preamplification (mIPEP), and multiple displacementamplification (MDA), are examples of whole genome amplification methods.

The term “short tandem repeat” or “STR” as used herein refers to a classof polymorphisms that occurs when a pattern of two or more nucleotidesare repeated and the repeated sequences are directly adjacent to eachother. The pattern can range in length from 2 to 10 base pairs (bp) (forexample (CATG)n in a genomic region) and is typically in the non-codingintron region. By examining several STR loci and counting how manyrepeats of a specific STR sequence there are at a given locus, it ispossible to create a unique genetic profile of an individual.

The term “primer,” as used herein refers to an isolated oligonucleotidewhich is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer, use ofthe method, and the parameters used for primer design, as disclosedherein.

The term “primer pair” or “primer set” refers to a set of primersincluding a 5′ “upstream primer” or “forward primer” that hybridizeswith the complement of the 5′ end of the DNA sequence to be amplifiedand a 3′ “downstream primer” or “reverse primer” that hybridizes withthe 3′ end of the sequence to be amplified. As will be recognized bythose of skill in the art, the terms “upstream” and “downstream” or“forward” and “reverse” are not intended to be limiting, but ratherprovide illustrative orientation in particular embodiments. A primerpair is said to be “unique” if it can be employed to specificallyamplify a particular target nucleotide sequence in a given amplificationmixture.

A “polymorphic marker” or “polymorphic site” is a locus at whichnucleotide sequence divergence occurs. The locus may be as small as onebase pair. Illustrative markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more typically greaterthan 10% or 20% of a selected population. A polymorphic site may be assmall as one base pair. Polymorphic markers include restriction fragmentlength polymorphism (RFLPs), variable number of tandem repeats (VNTR's),hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,deletions, and insertion elements such as Alu. The first identifiedallelic form is arbitrarily designated as the reference form and otherallelic forms are designated as alternative or variant alleles. Theallelic form occurring most frequently in a selected population issometimes referred to as the wild type form. Diploid organisms may behomozygous or heterozygous for allelic forms. A diallelic polymorphismhas two forms. A triallelic polymorphism has three forms. A polymorphismbetween two nucleic acids can occur naturally, or be caused by exposureto or contact with chemicals, enzymes, or other agents, or exposure toagents that cause damage to nucleic acids, for example, ultravioletradiation, mutagens or carcinogens. The terms “polymorphic locus” and“polymorphic site” are herein used interchangeably.

The terms “polymorphic target nucleic acid”, “polymorphic sequence”,“polymorphic target nucleic acid sequence” and “polymorphic nucleicacid” are used interchangeably herein to refer to a nucleic acidsequence e.g. a DNA sequence, that comprises one or more polymorphicsites e.g one SNP or a tandem SNP. Polymorphic sequences according tothe present technology can be used to specifically differentiate betweenmaternal and non-maternal alleles in the maternal sample comprising amixture of fetal and maternal nucleic acids.

A “single nucleotide polymorphism” (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. SNPs canalso arise from a deletion of a nucleotide or an insertion of anucleotide relative to a reference allele. Single nucleotidepolymorphisms (SNPs) are positions at which two alternative bases occurat appreciable frequency (>1%) in the human population, and are the mostcommon type of human genetic variation.

As used herein, the term “short tandem repeat” or “STR” as used hereinrefers to a class of polymorphisms that occurs when a pattern of two ormore nucleotides are repeated and the repeated sequences are directlyadjacent to each other. The pattern can range in length from 2 to 10base pairs (bp) (for example (CATG)n in a genomic region) and istypically in the non-coding intron region. By examining several STR lociand counting how many repeats of a specific STR sequence there are at agiven locus, it is possible to create a unique genetic profile of anindividual.

As used herein, the term “miniSTR” herein refers to tandem repeat offour or more base pairs that spans less than about 300 base pairs, lessthan about 250 base airs, less than about 200 base pairs, less thanabout 150 base pairs, less than about 100 base pairs, less than about 50base pairs, or less than about 25 base pairs. “miniSTRs” are STRs thatare amplifiable from cIDNA templates.

The term “tandem SNPs” herein refers to two or more SNPs that arepresent within a polymorphic target nucleic acid sequence.

The terms “plurality of polymorphic target nucleic acids”, “polymorphicnucleic acids” and “polymorphic sequences” are used interchangeablyherein and refer to a number of nucleic acid sequences each comprisingat least one polymorphic site e.g. one SNP, such that at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 40 or more differentpolymorphic sites are amplified from the polymorphic target nucleicacids to identify and/or quantify fetal alleles present in maternalsamples comprising fetal and maternal nucleic acids.

As used herein, the term “substantially cell free” encompassespreparations of the desired sample from which components that arenormally associated with it are removed. For example, a plasma sample isrendered essentially cell free by removing blood cells e.g. red cells,that are normally associated with it. In some embodiments, substantiallyfree samples are processed to remove cells that would otherwisecontribute to the desired genetic material that is to be tested for anabnormality

As used herein the term “chromosome” refers to the heredity-bearing genecarrier of a living cell which is derived from chromatin and whichcomprises DNA and protein components (especially histones). Theconventional internationally recognized individual human genomechromosome numbering system is employed herein. The size of anindividual chromosome can vary from one type to another with a givenmulti-chromosomal genome and from one genome to another. In the case ofthe human genome, the entire DNA mass of a given chromosome is usuallygreater than about 100,000,000 bp. For example, the size of the entirehuman genome is about 3.times.10.sup.9 bp. The largest chromosome,chromosome no. 1, contains about 2.4.times.10.sup.8 by while thesmallest chromosome, chromosome no. 22, contains about5.3.times.10.sup.7 bp.

The term “oligonucleotide” is used to refer to a nucleic acid that isrelatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides, most particularly, shorterthan 50 nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but primers are typically at least 7nucleotides long and, more typically range from 10 to 30 nucleotides, oreven more typically from 15 to 30 nucleotides, in length. Other primerscan be somewhat longer, e.g., 30 to 50 nucleotides long.

The term “allele call” as used herein, refers to successfulcharacterization of an allele by a given analysis method. If theanalysis provides successful characterization of both alleles of a genelocus of a DNA sample, it is said that two allele calls are made. If oneallele is characterized while the other allele is not characterized, itis said that one allele call is made. If neither of the two alleles issuccessfully characterized, no allele calls are made.

The term “allele” as used herein, is any one of a number of viable DNAcodings occupying a given locus (position) on a chromosome. Usuallyalleles are DNA (deoxyribonucleic acid) sequences that code for a gene,but sometimes the term is used to refer to a non-gene sequence. Anindividual's genotype for that gene is the set of alleles it happens topossess. In a diploid organism, one that has two copies of eachchromosome, two alleles make up the individual's genotype.

The term “reaction mixture” as used herein refers to a mixturecontaining sufficient components to carry out an amplification reaction.

The term “sequence tag density” herein refers to the number of sequencereads that are mapped to a reference genome sequence e.g. the sequencetag density for chromosome 21 is the number of sequence reads generatedby the sequencing method that are mapped to chromosome 21 of thereference genome. The term “sequence tag density ratio” herein refers tothe ratio of the number of sequence tags that are mapped to a chromosomeof the reference genome e.g. chromosome 21, to the length of thereference genome chromosome 21.

The terms “threshold value” and “qualified threshold value” herein referto any number that is calculated using a qualifying data set and servesas a limit of diagnosis of a copy number variation e.g. an aneuploidy,in an organism. If a threshold is exceeded by results obtained frompracticing the invention, a subject can be diagnosed with a copy numbervariation e.g. trisomy 21.

The term “read” refers to a DNA sequence of sufficient length (e.g., atleast about 30 bp) that can be used to identify a larger sequence orregion, e.g. that can be aligned and specifically assigned to achromosome or genomic region or gene.

The term “sequence tag” is herein used interchangeably with the term“mapped sequence tag” to refer to a sequence read that has beenspecifically assigned i.e. mapped, to a larger sequence e.g. a referencegenome, by alignment. Mapped sequence tags are uniquely mapped to areference genome i.e. they are assigned to a single location to thereference genome. Tags that can be mapped to more than one location on areference genome i.e. tags that do not map uniquely, are not included inthe analysis.

The terms “aligned”, “alignment”, or “aligning” refer to one or moresequences that are identified as a match in terms of the order of theirnucleic acid molecules to a known sequence from a reference genome. Suchalignment can be done manually or by a computer algorithm, examplesincluding the Efficient Local Alignment of Nucleotide Data (ELAND)computer program distributed as part of the Illumina Genomics Analysispipeline. The matching of a sequence read in aligning can be a 100%sequence match or less than 100% (non-perfect match).

The term “reference genome” refers to any particular known genomesequence, whether partial or complete, of any organism or virus whichmay be used to reference identified sequences from a subject. Forexample, a reference genome used for human subjects as well as manyother organisms is found at the National Center for BiotechnologyInformation at www.ncbi.nlm.nih.gov. A “genome” refers to the completegenetic information of an organism or virus, expressed in nucleic acidsequences.

The term “artificial target sequences genome” herein refers to agrouping of known sequences that encompass alleles of known polymorphicsites. For example, a “SNP reference genome” is an artificial targetsequences genome comprising a grouping of sequences that encompassalleles of known SNPs.

The term “clinically-relevant sequence” herein refers to a nucleic acidsequence that is known or is suspected to be associated or implicatedwith a genetic or disease condition. Determining the absence or presenceof a clinically-relevant sequence can be useful in determining adiagnosis or confirming a diagnosis of a medical condition, or providinga prognosis for the development of a disease.

The term “mixed sample” herein refers to a sample containing a mixtureof nucleic acids, which are derived from different genomes.

The term “original maternal sample” herein refers to a biological sampleobtained from a pregnant subject e.g. a woman, who serves as the sourcefrom which a portion is removed to amplify polymorphic target nucleicacids. The “original sample” can be any sample obtained from a pregnantsubject, and the processed fractions thereof e.g. a purified cfDNAsample extracted from a maternal plasma sample. The term “originalmaternal sample” herein refers to a biological sample obtained from apregnant subject e.g. a woman, who serves as the source from which aportion is removed to amplify polymorphic target nucleic acids. The“original sample” can be any sample obtained from a pregnant subject,and the processed fractions thereof e.g. a purified cfDNA sampleextracted from a maternal plasma sample.

The term “biological fluid” herein refers to a liquid taken from abiological source and includes, for example, blood, serum, plasma,sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears,saliva, and the like. As used herein, the terms “blood,” “plasma” and“serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

The terms “maternal nucleic acids” and “fetal nucleic acids” hereinrefer to the nucleic acids of a pregnant female subject and the nucleicacids of the fetus being carried by the pregnant female, respectively.

The term “corresponding to” herein refers to a nucleic acid sequencee.g. a gene or a chromosome, that is present in the genome of differentsubjects, and which does not necessarily have the same sequence in allgenomes, but serves to provide the identity rather than the geneticinformation of a sequence of interest e.g. a gene or chromosome.

The term “group of chromosomes” herein refers to two or morechromosomes.

The term “subject” herein refers to a human subject as well as anon-human subject such as a mammal, an invertebrate, a vertebrate, afungus, a yeast, a bacteria, and a virus. Although the examples hereinconcern human cells and the language is primarily directed to humanconcerns, the concept of this invention is applicable to genomes fromany plant or animal, and is useful in the fields of veterinary medicine,animal sciences, research laboratories and such.

DESCRIPTION

The methods described herein enable the determination of the fraction ofthe minor fetal nucleic acid component in a sample comprising a mixtureof fetal and maternal nucleic acids. In particular, the method enablesthe determination of the fraction of cfDNA contributed by a fetus to themixture of fetal and maternal cfDNA in a maternal sample e.g. a plasmasample. The difference between the maternal and fetal fraction isdetermined by the relative contribution of a polymorphic allele derivedfrom the fetal genome to the contribution of the correspondingpolymorphic allele derived from the maternal genome. Polymorphicsequences can be used in conjunction with clinically-relevant diagnostictests as a positive control for the presence of cfDNA in order tohighlight false-negative or false-positive results stemming from lowlevels of cfDNA below the identification limit The methods described areindependent of the gender of the fetus, and are useful across a range ofgestational ages.

FIG. 1 provides a flow diagram of an embodiment of method of theinvention 100 for determining the fraction of fetal nucleic acids in amaternal biological sample by massively parallel sequencing ofPCR-amplified polymorphic target nucleic acids. In step 110 a maternalsample comprising a mixture of fetal and maternal nucleic acids isobtained from a subject. The sample is a maternal sample that isobtained from a pregnant female, for example a pregnant woman. Othermaternal samples can be from mammals, for example, cow, horse, dog, orcat. If the subject is a human, the sample can be taken in the first orsecond trimester of pregnancy. Any maternal biological sample can beused a source of fetal and maternal nucleic acids which are contained incells or that are “cell-free”. In some embodiments, it is advantageousto obtain a maternal sample that comprises cell-free nucleic acids e.g.cfDNA. Preferably, the maternal biological sample is a biological fluidsample. A biological fluid includes, as non-limiting examples, blood,plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph,saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginalflow, transcervical lavage, brain fluid, ascites, milk, secretions ofthe respiratory, intestinal and genitourinary tracts, and leukophoresissamples. In some embodiments, the biological fluid sample is a samplethat is easily obtainable by non-invasive procedures e.g. blood, plasma,serum, sweat, tears, sputum, urine, sputum, ear flow, and saliva. Insome embodiments, the biological sample is a peripheral blood sample, orthe plasma and/or the serum fractions thereof. In another embodiment,the sample is a mixture of two or more biological samples e.g. abiological sample can comprise two or more of a biological fluidsamples. As used herein, the terms “blood,” “plasma” and “serum”expressly encompass fractions or processed portions thereof. In someembodiments, the biological sample is processed to obtain a samplefraction e.g. plasma, that contains the mixture of fetal and maternalnucleic acids. A sample that can be used to determine the genotype ofone or more fetal alleles can be any sample that contains fetal cells orfetal nucleic acid. For example, maternal serum or plasma samplecomprising fetal and maternal cell-free nucleic acids (e.g., DNA or RNA)can be used to determine the genotypes of fetal alleles. In oneembodiment, the sample can comprise a fetal cell, e.g., a fetalnucleated red blood cell or a trophoblast.

In step 120, the mixture of fetal and maternal nucleic acids is furtherprocessed from the sample fraction e.g. plasma, to obtain a samplecomprising a purified mixture of fetal and maternal nucleic acids e.g.cIDNA. Cell-free nucleic acids, including cell-free DNA, can be obtainedby various methods known in the art from biological samples includingbut not limited to plasma, serum and urine (Fan et al., Proc Natl AcadSci 105:16266-16271 [2008]; Koide et al., Prenatal Diagnosis 25:604-607[2005]; Chen et al., Nature Med. 2: 1033-1035 [1996]; Lo et al., Lancet350: 485-487 [1997). To separate cIDNA from cells, fractionation,centrifugation (e.g., density gradient centrifugation), DNA-specificprecipitation, or high-throughput cell sorting and/or separation methodscan be used. Examples of methods for processing fluid samples have beenpreviously disclosed, e.g., U.S. Patent Application Nos. 20050282293,20050224351, and 20050065735, which are herein incorporated by referencein their entireties. Commercially available kits for manual andautomated separation of cfDNA are available (Roche Diagnostics,Indianapolis, Ind., Qiagen, Valencia, Calif., Macherey-Nagel, Duren,Del.). In some instances, it can be advantageous to fragment the nucleicacid molecules in the nucleic acid sample. Fragmentation can be random,or it can be specific, as achieved, for example, using restrictionendonuclease digestion. Methods for random fragmentation are well knownin the art, and include, for example, limited DNAse digestion, alkalitreatment and physical shearing. In one embodiment, sample nucleic acidsare obtained as cfDNA, which is not subjected to fragmentation. In otherembodiments, the sample nucleic acids are obtained as genomic DNA, whichis subjected to fragmentation into fragments of approximately 500 ormore base pairs, and to which NGS methods can be readily applied.

In step 130, a portion of the purified mixture of fetal and maternalcIDNA is used for amplifying a plurality of polymorphic target nucleicacids each comprising a polymorphic site. In some embodiments, thetarget nucleic acids each comprise a SNP. In other embodiments, each ofthe target nucleic acids comprises a pair of tandem SNPs. In yet otherembodiments, each the target nucleic acids comprises an STR. Polymorphicsites that are contained in the target nucleic acids include withoutlimitation single nucleotide polymorphisms (SNPs), tandem SNPs,small-scale multi-base deletions or insertions, called IN-DELS (alsocalled deletion insertion polymorphisms or DIPs), Multi-NucleotidePolymorphisms (MNPs) Short Tandem Repeats (STRs), restriction fragmentlength polymorphism (RFLP), or a polymorphism comprising any otherchange of sequence in a chromosome. In some embodiments, the polymorphicsites that are encompassed by the method of the invention are located onautosomal chromosomes, thereby enabling the determination of fetalfraction independently of sex of the fetus. Polymorphisms associatedwith chromosomes other than chromosome 13, 18, 21 and Y can also be usedin the methods described herein.

Polymorphisms can be indicative, informative, or both. Indicativepolymorphisms indicate the presence of fetal cell-free DNA in a maternalsample. For example, the more there is of a particular genetic sequence,e.g. a SNP, the more a method will translate its presence into aparticular color intensity, density of color, or some other propertywhich is detectable and measurable and indicative of the presence,absence, and quantity of a particular fragment of DNA and/or particularpolymorphism e.g. SNP of the embryo. Informative polymorphisms yieldinformation about the fetus—for example, the presence or absence of adisease, genetic abnormality, or any other biological information suchas the stage of gestation or gender. With regard to the presentinvention, the methods are not conducted using all possible SNPs in agenome, but use those which are said to be “informative”. “InformativeSNPs” in this instance are those which identify differences in thesequence of the mother and the fetus. Any polymorphic site that can beencompassed by the reads generated by the sequencing methods describedherein can be used to determine the fetal fraction.

In one embodiment, a portion of the mixture of fetal and maternalnucleic acids in the sample e.g. cfDNA, is used as template foramplifying target nucleic acids that comprise at least one SNP. In someembodiments, each target nucleic acid comprises a single i.e. one SNP.Target nucleic acid sequences comprising SNPs are available frompublicly accessible databases including, but not limited to Human SNPDatabase at world wide web address wi.mit.edu, NCBI dbSNP Home Page atworld wide web address ncbi.nlm nih.gov, world wide web addresslifesciences.perkinelmer.com, Applied Biosystems by Life Technologies™(Carlsbad, Calif.) at world wide web address appliedbiosystems.com,Celera Human SNP database at world wide web address celera.com, the SNPDatabase of the Genome Analysis Group (GAN) at world wide web addressgan.iarc.fr. In one embodiment, the SNPs chosen for enriching the fetaland maternal cfDNA are selected from the group of 92 individualidentification SNPs (IISNPs) described by Pakstis el al. (Pakstis et el.Hum Genet 127:315-324 [2010]), which have been shown to have a verysmall variation in frequency across populations (F_(st)<0.06), and to behighly informative around the world having an average heterozygosity≧0.4. SNPs that are encompassed by the method of the invention includelinked and unlinked SNPs. Other useful SNPs applicable or useful for themethods described herein are disclosed in U.S. Pat. Application Nos.20080070792, 20090280492, 20080113358, 20080026390, 20080050739,20080220422, and 20080138809, which are herein incorporated by referencein their entireties. Each target nucleic acid comprises at least onepolymorphic site e.g. a single SNP, that differs from that present onanother target nucleic acid to generate a panel of polymorphic sitese.g. SNPs, that contain a sufficient number of polymorphic sites ofwhich at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 25, at least30, at least 40 or more are informative. For example, a panel of SNPscan be configured to comprise at least one informative SNP. In oneembodiment, the SNPs that are targeted for amplification are selectedfrom rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56). In one embodiment, the panel ofSNPs comprises at least 3, at least 5, at least 10, at least 13, atleast 15, at least 20, at least 25, at least 30 or more SNPs. In oneembodiment, the panel of SNPs comprises rs560681 (SEQ ID NOS 1 & 2),rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883(SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ IDNOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 &16), rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20),rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), andrs2567608 (SEQ ID NOS 25 & 26). The polymorphic nucleic acids comprisingthe SNPs can be amplified using exemplary primer pairs provided inExample 3, and disclosed as SEQ ID NOs:57-112.

In other embodiments, each target nucleic acid comprises two or moreSNPs i.e. each target nucleic acid comprises tandem SNPs. Preferably,each target nucleic acid comprises two tandem SNPs. The tandem SNPs areanalyzed as a single unit as short haplotypes, and are provided hereinas sets of two SNPs. To identify suitable tandem SNP sequences, theInternational HapMap Consortium database can be searched (TheInternational HapMap Project, Nature 426:789-796 [2003]). The databaseis available on the world wide web at hapmap.org. In one embodiment,tandem SNPs that are targeted for amplification are selected from thefollowing sets of tandem pairs of SNPS rs7277033-rs2110153 (SEQ ID NOS312 & 313); rs2822654-rs1882882 (SEQ ID NOS 314 & 315);rs368657-rs376635 (SEQ ID NOS 316 & 317); rs2822731-rs2822732 (SEQ IDNOS 318 & 319); rs1475881-rs7275487 (SEQ ID NOS 320 & 321);rs1735976-rs2827016 (SEQ ID NOS 322 & 323); rs447340-rs2824097 (SEQ IDNOS 324 & 325); rs418989-rs13047336 (SEQ ID NOS 326 & 327);rs987980-rs987981 (SEQ ID NOS 328 & 329); rs4143392-rs4143391 (SEQ IDNOS 330 & 331); rs1691324-rs13050434 (SEQ ID NOS 332 & 333);rs11909758-rs9980111 (SEQ ID NOS 334 & 335); rs2826842-rs232414 (SEQ IDNOS 336 & 337); rs1980969-rs1980970 (SEQ ID NOS 338 & 339);rs9978999-rs9979175 (SEQ ID NOS 340 & 341); rs1034346-rs12481852 (SEQ IDNOS 342 & 343); rs7509629-rs2828358 (SEQ ID NOS 344 & 345);rs4817013-rs7277036 (SEQ ID NOS 346 & 347); rs9981121-rs2829696 (SEQ IDNOS 348 & 349); rs455921-rs2898102 (SEQ ID NOS 350 & 351);rs2898102-rs458848 (SEQ ID NOS 352 & 353); rs961301-rs2830208 (SEQ IDNOS 354 & 355); rs2174536-rs458076 (SEQ ID NOS 356 & 357);rs11088023-rs11088024 (SEQ ID NOS 358 & 359); rs1011734-rs1011733 (SEQID NOS 360 & 361); rs2831244-rs9789838 (SEQ ID NOS 362 & 363);rs8132769-rs2831440 (SEQ ID NOS 364 & 365); rs8134080-rs2831524 (SEQ IDNOS 366 & 367); rs4817219-rs4817220 (SEQ ID NOS 368 & 369);rs2250911-rs2250997 (SEQ ID NOS 370 & 371); rs2831899-rs2831900 (SEQ IDNOS 372 & 373); rs2831902-rs2831903 (SEQ ID NOS 374 & 375);rs11088086-rs2251447 (SEQ ID NOS 376 & 377); rs2832040-rs11088088 (SEQID NOS 378 & 379); rs2832141-rs2246777 (SEQ ID NOS 380 & 381);rs2832959-rs9980934 (SEQ ID NOS 382 & 383); rs2833734-rs2833735 (SEQ IDNOS 384 & 385); rs933121-rs933122 (SEQ ID NOS 386 & 387);rs2834140-rs12626953 (SEQ ID NOS 388 & 389); rs2834485-rs3453 (SEQ IDNOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS 392 & 393);rs2776266-rs2835001 (SEQ ID NOS 394 & 395); rs1984014-rs1984015 (SEQ IDNOS 396 & 397); rs7281674-rs2835316 (SEQ ID NOS 398 & 399);rs13047304-rs13047322 (SEQ ID NOS 400 & 401); rs2835545-rs4816551 (SEQID NOS 402 & 403); rs2835735-rs2835736 (SEQ ID NOS 404 & 405);rs13047608-rs2835826 (SEQ ID NOS 406 & 407); rs2836550-rs2212596 (SEQ IDNOS 408 & 409); rs2836660-rs2836661 (SEQ ID NOS 410 & 411);rs465612-rs8131220 (SEQ ID NOS 412 & 413); rs9980072-rs8130031 (SEQ IDNOS 414 & 415); rs418359-rs2836926 (SEQ ID NOS 416 & 417);rs7278447-rs7278858 (SEQ ID NOS 418 & 419); rs385787-rs367001 (SEQ IDNOS 420 & 421); rs367001-rs386095 (SEQ ID NOS 422 & 423);rs2837296-rs2837297 (SEQ ID NOS 424 & 425); and rs2837381-rs4816672 (SEQID NOS 426 & 427). The polymorphic nucleic acids comprising the tandemSNPs can be amplified using primer pairs that amplify polymorphicsequences comprising the tandem SNPs. Examples of primer pairs that canbe used to amplify the tandem SNPs disclosed herein are SEQ IDNOs:197-310 as provided in Example 8.

In one embodiment, a portion of the mixture of fetal and maternalnucleic acids in the sample e.g. cfDNA, is used as template foramplifying target nucleic acids that comprise at least one STR. In someembodiments, each target nucleic acid comprises a single i.e. one STR.STR loci are found on almost every chromosome in the genome and may beamplified using a variety of polymerase chain reaction (PCR) primers.Tetranucleotide repeats have been preferred among forensic scientistsdue to their fidelity in PCR amplification, although some tri- andpentanucleotide repeats are also in use. A comprehensive listing ofreferences, facts and sequence information on STRs, published PCRprimers, common multiplex systems, and related population data arecompiled in STRBase, which may be accessed via the World Wide Web atibm4.carb.nist.gov:8800/dna/home.htm. Sequence information from GenBank®(http://www2.ncbi.nlm nih.gov/cgi-bin/genbank) for commonly used STRloci is also accessible through STRBase. Commercial kits available forthe analysis of STR loci generally provide all of the necessary reactioncomponents and controls required for amplification. STR multiplexsystems allow the simultaneous amplification of multiple nonoverlappingloci in a single reaction, substantially increasing throughput. Withmulticolor fluorescent detection, even overlapping loci can bemultiplexed. The polymorphic nature of tandem repeated DNA sequencesthat are widespread throughout the human genome have made them importantgenetic markers for gene mapping studies, linkage analysis, and humanidentity testing. Because of the high polymorphism of STRs, mostindividuals will be heterozygous i.e. most people will possess twoalleles (versions) of each—one inherited from each parent with adifferent number of repeats. The PCR products comprising the STRs can beseparated and detected using manual, semi-automated or automatedmethods. Semi-automated systems are gel-based and combineelectrophoresis, detection and analysis into one unit. On asemiautomated system, gel assembly and sample loading are still manualprocesses; however, once samples are loaded onto the gel,electrophoresis, detection and analysis proceed automatically. Datacollection occurs in “real time” as fluorescently labeled fragmentsmigrate past the detector at a fixed point and can be viewed as they arecollected. As the name implies, capillary electrophoresis is carried outin a microcapillary tube rather than between glass plates. Once samples,gel polymer and buffer are loaded onto the instrument, the capillary isfilled with gel polymer and the sample is loaded automatically.Therefore, the non-maternally inherited fetal STR sequence will differin the number of repeats from the maternal sequence. Amplification ofthese STR sequences can result in one or two major amplificationproducts corresponding to the maternal alleles (and the maternallyinherited fetal allele) and one minor product corresponding to thenon-maternally inherited fetal allele. This technique was first reportedin 2000 (Pertl et al., Human Genetics 106:45-49 [2002]) and hassubsequently been developed using simultaneous identification ofmultiple different STR regions using real-time PCR (Liu et al., ActaObset Gyn Scand 86:535-541 [2007]). Various sized PCR amplicons havebeen used to discern the respective size distributions of circulatingfetal and maternal DNA species, and have shown that the fetal DNAmolecules in the plasma of pregnant women are generally shorter thanmaternal DNA molecules (Chan et al., Clin Chem 50:8892 [2004]). Sizefractionation of circulating fetal DNA has confirmed that the averagelength of circulating fetal DNA fragments is <300 bp, while maternal DNAhas been estimated to be between about 0.5 and 1 Kb (Li et al., ClinChem, 50: 1002-1011 [2004]). The invention provides a method fordetermining the fraction of fetal nucleic acid in a maternal samplecomprising determining the amount of copies of at least one fetal andone maternal allele at a polymorphic miniSTR site, which can beamplified to generate amplicons that are of lengths about the size ofthe circulating fetal DNA fragments e.g. less than about 250 base pairs.In one embodiment, the fetal fraction can be determined by a method thatcomprises sequencing at least a portion of amplified polymorphic targetnucleic acids each comprising a miniSTR. Fetal and maternal alleles atan informative STR site are discerned by their different lengths i.e.number of repeats, and the fetal fraction can be calculated as a percentratio of the amount of fetal maternal alleles at that site. The methodcan use one or a combination of any number of informative miniSTRs todetermine the fraction of fetal nucleic acid. For example, any one or acombination of any number of miniSTRs, for example the miniSTRsdisclosed in Table 7 and FIGS. 4 and 5, can be used. In one embodiment,the fraction of fetal nucleic acid in a maternal sample is performedusing a method that includes determining the number of copies of thematernal and fetal nucleic acid present in the maternal sample byamplifying at least one autosomal miniSTR chosen from CSF1PO, FGA, TH01,TPDX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51,D21S11, Penta D, Penta E, D2S1338, D1S1677, D2S441, D4S2364, D10S1248,D14S1434, D22S1045, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301,D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157,D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. Inanother embodiment, the at least one autosomal miniSTR is the group ofminiSTRs CSF1PO, FGA, D13S317, D16S539, D18S51, D2S1338, D21S11, D2S1338and D7S820. In one embodiment, the method comprises determining thenumber of copies of at least one fetal and at least one maternal alleleat least at one polymorphic miniSTR that is amplified to generateamplicons that are less than about 300 bp, less than about 250 bp, lessthan about 200 bp, less than about 150 bp, less than about 100 bp, orless than about 50 bp. In another embodiment, the amplicons that aregenerated by amplifying the miniSTRs are less than about 300 bp. Inanother embodiment, the amplicons that are generated by amplifying theminiSTRs are less than about 250 bp. In another embodiment, theamplicons that are generated by amplifying the miniSTRs are less thanabout 200 bp. Amplification of the informative allele includes usingminiSTR primers, which allow for the amplification of reduced-sizeamplicons to detect STR alleles that are less than about 500 bp, lessthan about 450 bp, less than about 400 bp, less than about 350 bp, lessthan about 300 base pairs (bp), less than about 250 bp, less than about200 bp, less than about 150 bp, less than about 100 bp, or less thanabout 50 bp. The reduced-size amplicons generated using the miniSTRprimers are known as miniSTRs that are identified according to themarker name corresponding to the locus to which they have been mapped.In one embodiment, the miniSTR primers include mini STR primers thathave permitted the maximum size reduction in amplicon size for all 13CODIS STR loci in addition to the D2S1338, Penta D, and pentaE found incommercially available STR kits (Butler et al., J Forensic Sci48:1054-1064 [2003]), miniSTR loci that are unlinked to the CODISmarkers as described by Coble and Butler (Coble and Butler, J ForensicSci 50:43-53 [2005]), and other minSTRs that have been characterized atNIST. Information regarding the miniSTRs characterized at NIST isaccessible via the world wide web atcstl.nist.gov/biotech/strbase/newSTRs.htm. Any one pair or a combinationof two or more pairs of miniSTR primers can be used to amplify at leastone miniSTR.

In one embodiment, exemplary primer sets that can be used to amplifySTRs in maternal cIDNA samples include the primer sets provided inExample 9 and disclosed as SEQ ID NOs:113-196.

Gender identification (sex-typing) in commonly performed in conjunctionwith STR typing using PCR products generated from the Amelogenin genethat occurs on both the X- and Y-chromosome. Amelogenin is not an STRlocus, but it produces X and Y chromosome specific PCR products. Acommonly used PCR primer set first published by Sullivan et al. (1993)(Sullivan et al., BioTechniques 15:637-641 [1993]) targets a 6 bydeletion that occurs on the X-chromosome, which enables ampliconsgenerated from the X- and Y-chromosome to be distinguished from oneanother when electrophoretic separation is performed to separate STRalleles. Most commercial STR kits utilize the Sullivan et al. (1993)primers or minor modifications. Since females are X,X, only a singlepeak is observed when testing female DNA whereas males, which possessboth X and Y chromosomes, exhibit two peaks with a standard Amelogenintest. In one embodiment, the method to determine the fraction of fetalnucleic acid in a maternal sample comprises coamplifying Ameleogeninwith at least one miniSTR. In another embodiment, the method does notcomprise coamplifying Amelogenin with miniSTR loci.

Amplification of the target nucleic acids in the mixture of fetal andmaternal nucleic acid e.g. cfDNA, is accomplished any method that usesPCR or variations of the method including but not limited to digitalPCR, real time PCR (RT-PCR), TaqMan PCR System (Applied Biosystems),SNPlex or GenPlex methods, asymmetric PCR, helicase-dependentamplification, hot-start PCR, qPCR, solid phase PCR, and touchdown PCR.Alternatively, replication of target nucleic acid sequences can beobtained by enzyme-independent methods e.g. chemical solid-phasesynthesis using the phosphoramidites. Amplification of the targetsequences is accomplished using primer pairs each capable of amplifyinga target nucleic acid sequence comprising the polymorphic site e.g. SNP,in a multiplex PCR reaction. Multiplex PCR reactions include combiningat least 2, at least three, at least 3, at least 5, at least 10, atleast 15, at least 20, at least 25, at least 30, at least 40 or moresets of primers in the same reaction to quantify the amplified targetnucleic acids comprising at least two, at least three, at least 5, atleast 10, at least 15, at least 20, at least 25, at least 30, at least40 or more polymorphic sites in the same sequencing reaction. Any panelof primer sets can be configured to amplify at least one informativepolymorphic sequence.

In step 140 of method 100 (FIG. 1), a portion or all of the amplifiedpolymorphic sequences are used to prepare a sequencing library forsequencing in a parallel fashion as described. In one embodiment, thelibrary is prepared for sequencing-by-synthesis using Illumina'sreversible terminator-based sequencing chemistry.

In step 140, sequence information that is needed for determining fetalfraction is obtained using any of the known DNA sequencing methods. Inone embodiment, the method described herein employs next generationsequencing technology (NGS) in which clonally amplified DNA templates orsingle DNA molecules are sequenced in a massively parallel fashionwithin a flow cell (e.g. as described in Volkerding et al. Clin Chem55:641-658 [2009]; Metzker M Nature Rev 11:31-46 [2010]). In addition tohigh-throughput sequence information, NGS provides digital quantitativeinformation, in that each sequence read is a countable “sequence tag”representing an individual clonal DNA template or a single DNA molecule.This quantification allows NGS to expand the digital PCR concept ofcounting cell-free DNA molecules (Fan et al., Proc Natl Acad Sci USA105:16266-16271 [2008]; Chiu et al., Proc Natl Acad Sci USA 2008;105:20458-20463 [2008]). The sequencing technologies of NGS includepyrosequencing, sequencing-by-synthesis with reversible dye terminators,sequencing by oligonucleotide probe ligation and real time sequencing.

Some of the sequencing technologies are available commercially, such asthe sequencing-by-hybridization platform from Affymetrix Inc.(Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) andHelicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligationplatform from Applied Biosystems (Foster City, Calif.), as describedbelow. In addition to the single molecule sequencing performed usingsequencing-by-synthesis of Helicos Biosciences, other single moleculesequencing technologies are encompassed by the method of the inventionand include the SMRT™ technology of Pacific Biosciences, the Ion Torrenttechnology, and nanopore sequencing being developed for example, byOxford Nanopore Technologies.

While the automated Sanger method is considered as a ‘first generation’technology, Sanger sequencing including the automated Sanger sequencing,can also be employed by the method of the invention. Additionalsequencing methods that comprise the use of developing nucleic acidimaging technologies e.g. atomic force microscopy (AFM) or transmissionelectron microscopy (TEM), are also encompassed by the method of theinvention. Exemplary sequencing technologies are described below.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the Helicos True Single Molecule Sequencing(tSMS) (e.g. as described in Harris T. D. et al., Science 320:106-109[2008]). In the tSMS technique, a DNA sample is cleaved into strands ofapproximately 100 to 200 nucleotides, and a polyA sequence is added tothe 3′ end of each DNA strand. Each strand is labeled by the addition ofa fluorescently labeled adenosine nucleotide. The DNA strands are thenhybridized to a flow cell, which contains millions of oligo-T capturesites that are immobilized to the flow cell surface. The templates canbe at a density of about 100 million templates/cm². The flow cell isthen loaded into an instrument, e.g., HeliScope™ sequencer, and a laserilluminates the surface of the flow cell, revealing the position of eachtemplate. A CCD camera can map the position of the templates on the flowcell surface. The template fluorescent label is then cleaved and washedaway. The sequencing reaction begins by introducing a DNA polymerase anda fluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are discerned by imaging theflow cell surface. After imaging, a cleavage step removes thefluorescent label, and the process is repeated with other fluorescentlylabeled nucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the 454 sequencing (Roche) (e.g. as describedin Margulies, M. et al. Nature 437:376-380 [2005]). 454 sequencinginvolves two steps. In the first step, DNA is sheared into fragments ofapproximately 300-800 base pairs, and the fragments are blunt-ended.Oligonucleotide adaptors are then ligated to the ends of the fragments.The adaptors serve as primers for amplification and sequencing of thefragments. The fragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (pico-liter sized). Pyrosequencing isperformed on each DNA fragment in parallel. Addition of one or morenucleotides generates a light signal that is recorded by a CCD camera ina sequencing instrument. The signal strength is proportional to thenumber of nucleotides incorporated. Pyrosequencing makes use ofpyrophosphate (PPi) which is released upon nucleotide addition. PPi isconverted to ATP by ATP sulfurylase in the presence of adenosine 5′phosphosulfate. Luciferase uses ATP to convert luciferin tooxyluciferin, and this reaction generates light that is discerned andanalyzed.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the SOLiD technology (Applied Biosystems). InSOLiD sequencing-by-ligation, genomic DNA is sheared into fragments, andadaptors are attached to the 5′ and 3′ ends of the fragments to generatea fragment library. Alternatively, internal adaptors can be introducedby ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the single molecule, real-time (SMRT™)sequencing technology of Pacific Biosciences. In SMRT sequencing, thecontinuous incorporation of dye-labeled nucleotides is imaged during DNAsynthesis. Single DNA polymerase molecules are attached to the bottomsurface of individual zero-mode wavelength identifiers (ZMW identifiers)that obtain sequence information while phospholinked nucleotides arebeing incorporated into the growing primer strand. A ZMW is aconfinement structure which enables observation of incorporation of asingle nucleotide by DNA polymerase against the background offluorescent nucleotides that rapidly diffuse in an out of the ZMW (inmicroseconds). It takes several milliseconds to incorporate a nucleotideinto a growing strand. During this time, the fluorescent label isexcited and produces a fluorescent signal, and the fluorescent tag iscleaved off. Identification of the corresponding fluorescence of the dyeindicates which base was incorporated. The process is repeated.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is nanopore sequencing (e.g. as described inSoni G V and Meller A. Clin Chem 53: 1996-2001 [2007]). Nanoporesequencing DNA analysis techniques are being industrially developed by anumber of companies, including Oxford Nanopore Technologies (Oxford,United Kingdom). Nanopore sequencing is a single-molecule sequencingtechnology whereby a single molecule of DNA is sequenced directly as itpasses through a nanopore. A nanopore is a small hole, of the order of 1nanometer in diameter. Immersion of a nanopore in a conducting fluid andapplication of a potential (voltage) across it results in a slightelectrical current due to conduction of ions through the nanopore. Theamount of current which flows is sensitive to the size and shape of thenanopore. As a DNA molecule passes through a nanopore, each nucleotideon the DNA molecule obstructs the nanopore to a different degree,changing the magnitude of the current through the nanopore in differentdegrees. Thus, this change in the current as the DNA molecule passesthrough the nanopore represents a reading of the DNA sequence.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the chemical-sensitive field effecttransistor (chemFET) array (e.g., as described in U.S. PatentApplication Publication No. 20090026082). In one example of thetechnique, DNA molecules can be placed into reaction chambers, and thetemplate molecules can be hybridized to a sequencing primer bound to apolymerase. Incorporation of one or more triphosphates into a newnucleic acid strand at the 3′ end of the sequencing primer can bediscerned by a change in current by a chemFET. An array can havemultiple chemFET sensors. In another example, single nucleic acids canbe attached to beads, and the nucleic acids can be amplified on thebead, and the individual beads can be transferred to individual reactionchambers on a chemFET array, with each chamber having a chemFET sensor,and the nucleic acids can be sequenced.

In one embodiment, the DNA sequencing technology that is used in themethod of the invention is the Halcyon Molecular's method that usestransmission electron microscopy (TEM). The method, termed IndividualMolecule Placement Rapid Nano Transfer (IMPRNT), comprises utilizingsingle atom resolution transmission electron microscope imaging ofhigh-molecular weight (150 kb or greater) DNA selectively labeled withheavy atom markers and arranging these molecules on ultra-thin films inultra-dense (3 nm strand-to-strand) parallel arrays with consistentbase-to-base spacing. The electron microscope is used to image themolecules on the films to determine the position of the heavy atommarkers and to extract base sequence information from the DNA. Themethod is further described in PCT patent publication WO 2009/046445.The method allows for sequencing complete human genomes in less than tenminutes.

In one embodiment, the DNA sequencing technology is the Ion Torrentsingle molecule sequencing, which pairs semiconductor technology with asimple sequencing chemistry to directly translate chemically encodedinformation (A, C, G, T) into digital information (0, 1) on asemiconductor chip. In nature, when a nucleotide is incorporated into astrand of DNA by a polymerase, a hydrogen ion is released as abyproduct. Ion Torrent uses a high-density array of micro-machined wellsto perform this biochemical process in a massively parallel way. Eachwell holds a different DNA molecule. Beneath the wells is anion-sensitive layer and beneath that an ion sensor. When a nucleotide,for example a C, is added to a DNA template and is then incorporatedinto a strand of DNA, a hydrogen ion will be released. The charge fromthat ion will change the pH of the solution, which can be identified byIon Torrent's ion sensor. The sequencer essentially the world's smallestsolid-state pH meter—calls the base, going directly from chemicalinformation to digital information. The Ion personal Genome Machine(PGM™) sequencer then sequentially floods the chip with one nucleotideafter another. If the next nucleotide that floods the chip is not amatch. No voltage change will be recorded and no base will be called. Ifthere are two identical bases on the DNA strand, the voltage will bedouble, and the chip will record two identical bases called. Directidentification allows recordation of nucleotide incorporation inseconds.

Other sequencing methods include digital PCR and sequencing byhybridization. Digital polymerase chain reaction (digital PCR or dPCR)can be used to directly identify and quantify nucleic acids in a sample.Digital PCR can be performed in an emulsion. Individual nucleic acidsare separated, e.g., in a microfluidic chamber device, and each nucleiccan is individually amplified by PCR. Nucleic acids can be separatedsuch there is an average of approximately 0.5 nucleic acids/well, or notmore than one nucleic acid/well. Different probes can be used todistinguish fetal alleles and maternal alleles. Alleles can beenumerated to determine copy number. In sequencing by hybridization, thehybridization comprises contacting the plurality of polynucleotidesequences with a plurality of polynucleotide probes, wherein each of theplurality of polynucleotide probes can be optionally tethered to asubstrate. The substrate might be flat surface comprising an array ofknown nucleotide sequences. The pattern of hybridization to the arraycan be used to determine the polynucleotide sequences present in thesample. In other embodiments, each probe is tethered to a bead, e.g., amagnetic bead or the like. Hybridization to the beads can be identifiedand used to identify the plurality of polynucleotide sequences withinthe sample.

In one embodiment, the method employs massively parallel sequencing ofmillions of DNA fragments using Illumina's sequencing-by-synthesis andreversible terminator-based sequencing chemistry (e.g. as described inBentley et al., Nature 6:53-59 [2009]). Template DNA can be genomic DNAe.g. cfDNA. In some embodiments, genomic DNA from isolated cells is usedas the template, and it is fragmented into lengths of several hundredbase pairs. In other embodiments, cfDNA is used as the template, andfragmentation is not required as cIDNA exists as short fragments. Forexample fetal cIDNA circulates in the bloodstream as fragments of <300bp, and maternal cfDNA has been estimated to circulate as fragments ofbetween about 0.5 and 1 Kb (Li et al., Clin Chem, 50: 1002-1011 [2004]).Illumina's sequencing technology relies on the attachment of fragmentedgenomic DNA to a planar, optically transparent surface on whicholigonucleotide anchors are bound. Template DNA is end-repaired togenerate 5′-phosphorylated blunt ends, and the polymerase activity ofKlenow fragment is used to add a single A base to the 3′ end of theblunt phosphorylated DNA fragments. This addition prepares the DNAfragments for ligation to oligonucleotide adapters, which have anoverhang of a single T base at their 3′ end to increase ligationefficiency. The adapter oligonucleotides are complementary to theflow-cell anchors. Under limiting-dilution conditions, adapter-modified,single-stranded template DNA is added to the flow cell and immobilizedby hybridization to the anchors. Attached DNA fragments are extended andbridge amplified to create an ultra-high density sequencing flow cellwith hundreds of millions of clusters, each containing ˜1,000 copies ofthe same template. The cluster amplified DNA molecules are sequencedusing a robust four-color DNA sequencing-by-synthesis technology thatemploys reversible terminators with removable fluorescent dyes.High-sensitivity fluorescence identification is achieved using laserexcitation and total internal reflection optics. Short sequence reads ofabout 20-40 by e.g. 36 bp, are aligned against a repeat-masked referencegenome and genetic differences are called using specially developed dataanalysis pipeline software. After completion of the first read, thetemplates can be regenerated in situ to enable a second read from theopposite end of the fragments. Thus, either single-end or paired endsequencing of the DNA fragments is used according to the method. Partialsequencing of DNA fragments present in the sample is performed, andsequence tags comprising reads of predetermined length e.g. 36 bp, thatare mapped to a known reference genome are counted.

The length of the sequence read is associated with the particularsequencing technology. NGS methods provide sequence reads that vary insize from tens to hundreds of base pairs. In some embodiments of themethod described herein, the sequence reads are about 20 bp, about 25bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp,about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110bp, about 120 bp, about 130, about 140 bp, about 150 bp, about 200 bp,about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp,about 500 bp, about 550 bp or about 600 bp. It is expected thattechnological advances will enable single-end reads of greater than 500bp enabling for reads of greater than about 1000 bp when paired endreads are generated. In one embodiment, the sequence reads are 36 bp.Other sequencing methods that can be employed by the method of theinvention include the single molecule sequencing methods that cansequence nucleic acids molecules >5000 bp. The massive quantity ofsequence output is transferred by an analysis pipeline that transformsprimary imaging output from the sequencer into strings of bases. Apackage of integrated algorithms performs the core primary datatransformation steps: image analysis, intensity scoring, base calling,and alignment.

In one embodiment, partial sequencing of amplified target polymorphicnucleic acids is performed, and sequence tags comprising reads ofpredetermined length e.g. 36 bp, that map to a known reference genomeare counted. Only sequence reads that uniquely align to a referencegenome are counted as sequence tags. In one embodiment, the referencegenome is an artificial target sequences genome that comprises thesequences of the polymorphic target nucleic acids e.g. SNPs. In oneembodiment, the reference genome is an artificial SNP reference genome.In another r embodiment, the reference genome is an artificial STRreference genome. In yet another embodiment, the reference genome is anartificial tandem-STR reference genome. Artificial reference genomes canbe compiled using the sequences of the target polymorphic nucleic acids.Artificial reference genomes can comprise polymorphic target sequenceeach comprising one or more different types of polymorphic sequences.For example, an artificial reference genome can comprise polymorphicsequences comprising SNP alleles and/or STRs. In one embodiment, thereference genome is the human reference genome NCBI36/hg18 sequence,which is available on the world wide web atgenome.ucsc.edu/cgi-bin/hgGateway?org=Human&db=hg18&hgsid=166260105).Other sources of public sequence information include GenBank, dbEST,dbSTS, EMBL (the European Molecular Biology Laboratory), and the DDBJ(the DNA Databank of Japan). In another embodiment, the reference genomecomprises the human reference genome NCBI36/hg18 sequence and anartificial target sequences genome, which includes the targetpolymorphic sequences e.g. a SNP genome. Mapping of the sequence tags isachieved by comparing the sequence of the mapped tag with the sequenceof the reference genome to determine the chromosomal origin of thesequenced nucleic acid (e.g. cIDNA) molecule, and specific geneticsequence information is not needed. A number of computer algorithms areavailable for aligning sequences, including without limitation BLAST(Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993),FASTA (Person & Lipman, 1988), BOWTIE (Langmead et al., Genome Biology10:R25.1-R25.10 [2009]), or ELAND (Illumina, Inc., San Diego, Calif.,USA). In one embodiment, one end of the clonally expanded copies of theplasma cIDNA molecules is sequenced and processed by bioinformaticalignment analysis for the Illumina Genome Analyzer, which uses theEfficient Large-Scale Alignment of Nucleotide Databases (ELAND)software. In embodiments of the method that comprise determining thepresence or absence of an aneuploidy and fetal fraction using NGSsequencing methods, analysis of sequencing information for thedetermination of aneuploidy may allow for a small degree of mismatch(0-2 mismatches per sequence tag) to account for minor polymorphismsthat may exist between the reference genome and the genomes in the mixedsample. Analysis of sequencing information for the determination offetal fraction may allow for a small degree of mismatch depending on thepolymorphic sequence. For example, a small degree of mismatch may beallowed if the polymorphic sequence is an STR. In cases when thepolymorphic sequence is a SNP, all sequence that match exactly to eitherof the two alleles at the SNP site are counted first and filtered fromthe remaining reads, for which a small degree of mismatch may beallowed. Quantification of the number of sequence reads aligning to eachchromosome for determining chromosomal aneuploidies can be determined asdescribed herein, or using alternative analyses that employ normalizingthe median number of sequence tags for a chromosome of interest to themedian number of tags for each of the other autosomes (Fan et al., ProcNatl Acad Sci 105:16266-16271 [2008]), or that compare the number ofunique reads aligning to each chromosome to the total number of readsaligning to all chromosomes to derive a percent genomic representationfor each chromosome. A “z score” is generated to represent thedifference between the percent genomic representation of the chromosomeof interest and the mean percent representation for the same chromosomebetween a euploid control group, divided by the standard deviation (Chiuet al., Clin Chem 56:459-463 [2010]). In another embodiment, thesequencing information can be determined as described in U.S.Provisional Patent Application titled “Normalizing Biological Assays,”docket no. 32047-768.101, filed Jan. 19, 2010, which is hereinincorporated by reference in its entirety.

Analysis of sequencing information for the determination of fetalfraction may allow for a small degree of mismatch depending on thepolymorphic sequence. For example, a small degree of mismatch may beallowed if the polymorphic sequence is an STR. In cases when thepolymorphic sequence is a SNP, all sequences that match exactly toeither of the two alleles at the SNP site are counted first and filteredfrom the remaining reads, for which a small degree of mismatch may beallowed. The present method for determining fetal fraction by sequencingof nucleic acids can be used in combination with other methods.

In step 160, fetal fraction is determined based on the total number oftags that map to the first allele and the total number of tags that mapto second allele at an informative polymorphic site e.g. a SNP,contained in a reference genome. For example, the reference genome is anartificial target sequence genome that encompasses the polymorphicsequences that comprise SNPs rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 &8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12),rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16),rs4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20),rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), rs2567608(SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171 (SEQ IDNOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ ID NOS 33 &34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 & 38),rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42), rs508485(SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254 (SEQ IDNOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS 51 &52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 & 56).In one embodiment, the artificial reference genome includes thepolymorphic target sequences of SEQ ID NOs:1-56 (see Example-3).

In another embodiment, the artificial genome is an artificial targetsequence genome that encompasses polymorphic sequences that comprisetandem SNPs rs7277033-rs2110153 (SEQ ID NOS 312 & 313);rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ IDNOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319);rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ IDNOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325);rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ IDNOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331);rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337);rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ IDNOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343);rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ IDNOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349);rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ IDNOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355);rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ IDNOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361);rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ IDNOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367);rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ IDNOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373);rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ IDNOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379);rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ IDNOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385);rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ IDNOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391);rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ IDNOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397);rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403);rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ IDNOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409);rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ IDNOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415);rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ IDNOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421);rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ IDNOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).

In another embodiment, the artificial target genome encompassespolymorphic sequences that comprise STRs selected from CSF1PO, FGA,TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539,D18S51, D21S11, Penta D, Penta E, D2S1338, D1S1677, D2S441, D4S2364,D10S1248, D14S1434, D22S1045, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D5S2500, D4S2408,D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, andD1GATA113. The composition of the artificial target sequences genomewill vary depending on the polymorphic sequences that are used fordetermining the fetal fraction. Accordingly, an artificial targetsequences genome is not limited to the SNP, tandem SNP or STR sequencesexemplified herein.

The informative polymorphic site e.g. SNP, is identified by thedifference in the allelic sequences and the amount of each of thepossible alleles. Fetal cfDNA is present at a concentration that is <10%of the maternal cfDNA. Thus, the presence of a minor contribution of anallele to the mixture of fetal and maternal nucleic acids relative tothe major contribution of the maternal allele can be assigned to thefetus. Alleles that are derived from the maternal genome are hereinreferred to as major alleles, and alleles that are derived from thefetal genome are herein referred to as minor alleles. Alleles that arerepresented by similar levels of mapped sequence tags represent maternalalleles. The results of an exemplary multiplex amplification of targetnucleic acids comprising SNPs and derived from a maternal plasma sampleis shown in FIG. 2. Informative SNPs are discerned from the singlenucleotide change at a polymorphic site, and fetal alleles are discernedby their relative minor contribution to the mixture of fetal andmaternal nucleic acids in the sample when compared to the majorcontribution to the mixture by the maternal nucleic acids. Accordingly,the relative abundance of fetal cfDNA in the maternal sample isdetermined as a parameter of the total number of unique sequence tagsmapped to the target nucleic acid sequence on a reference genome foreach of the two alleles of the predetermined polymorphic site. In oneembodiment, the fraction of fetal nucleic acids in the mixture of fetaland maternal nucleic acids is calculated for each of the informativeallele (allele_(x)) as follows:

% fetal fraction allele_(x)=((ΣFetal sequence tags forallele_(x))/(ΣMaternal sequence tags for allele_(x)))×100 and fetalfraction for the sample is calculated as the average of the fetalfraction of all of the informative alleles. Optionally, the fraction offetal nucleic acids in the mixture of fetal and maternal nucleic acidsis calculated for each of the informative allele (allele_(x)) asfollows:

% fetal fraction allele_(x)=((2×ΣFetal sequence tags forallele_(x))/(ΣMaternal sequence tags for allele_(x)))×100,

to compensate for the presence of 2 fetal alleles, one being masked bythe maternal background.

The percent fetal fraction is calculated for at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 40 or more informativealleles. In one embodiment, the fetal fraction is the average fetalfraction determined for at least 3 informative alleles.

FIG. 3, shows a flowchart of alternate methods whereby fetal fractioncan be determined from amplified target nucleic acids that have beencombined with unamplified fetal and maternal cIDNA sample to allow forthe simultaneous determination of fetal fraction and the presence orabsence of fetal aneuploidy by enriching the maternal sample comprisingfetal and maternal nucleic acids for polymorphic target nucleic acids.In one embodiment, the sample that is enriched is the plasma fraction ofa blood sample (a). For example, a portion of an original maternalplasma sample is used for amplifying target nucleic acid sequences.Subsequently, some or all of the amplified product is combined with theremaining unamplified original plasma sample thereby enriching it (seeExample 7). In another embodiment, the sample that is enriched is thesample of purified cfDNA that is extracted from plasma (b). For example,enrichment comprises amplifying the target nucleic acids that arecontained in a portion of an original sample of purified mixture offetal and maternal nucleic acids e.g. cfDNA that has been purified froma maternal plasma sample, and subsequently combining some or all of theamplified product with the remaining unamplified original purifiedsample (see Example 6). In yet another embodiment, the sample that isenriched is a sequencing library sample prepared from a purified mixtureof fetal and maternal nucleic acids (c). For example, enrichmentcomprises amplifying the target nucleic acids that are contained in aportion of an original sample of purified mixture of fetal and maternalnucleic acids e.g. cfDNA that has been purified from a maternal plasmasample, preparing a first sequencing library of unamplified nucleic acidsequences, preparing a second sequencing library of amplifiedpolymorphic target nucleic acids, and subsequently combining some or allof the second sequencing library with some or all of the firstsequencing library (see Example 5). The amount of amplified product thatis used to enrich the original sample is selected to obtain sufficientsequencing information for determining both the presence or absence ofaneuploidy and the fetal fraction from the same sequencing run. At leastabout 3%, at least about 5%, at least about 7%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30% or more of the total number of sequence tags obtained fromsequencing are mapped to determine the fetal fraction. Sequencing of thelibrary generated following any one of the methods depicted in FIG. 3,provides sequence tags derived from the amplified target nucleic acidsand tags derived from the original unamplified maternal sample. Fetalfraction is calculated from the number of tags mapped to an artificialreference genome, and the presence or absence of aneuploidy isdetermined from the number of tags that map to the subject genome e.g.human genome.

An alternative method for determining fetal fraction from amplifiedpolymorphic target nucleic acids at step 130 of FIG. 1, uses sizeseparation of amplified polymorphic target nucleic acids comprising STRs(step 150 of FIG. 1). As described above, the polymorphic character of aSTR locus is due to variation in the number of tandemly repeated unitsbetween alleles. Because of the high polymorphism of the STRs mostindividuals will be heterozygous for STRs. Amplification of an STR willresult in one or two PCR products in most samples. In samples obtainedfrom pregnant women e.g. plasma samples, amplification of an STR willresult in one or two major PCR products, which correspond to the one ortwo maternal alleles including one fetal maternally-inherited allele,and a third paternally-inherited fetal allele that is detected at aninformative STR.

The STRs that are targeted for amplification are miniSTRs as describedherein that are less than about 300 base pairs and that are amplified ina multiplex PCR reaction, which allows the simultaneous amplification ofmultiple loci in a single reaction. The primers are labeled withdifferent fluorescent dyes each emitting fluorescence at a differentwavelength e.g. 6FAM™, VIC™, NED™, and PET™, and the number of repeatunits for each fluorescently tagged STR in the resulting PCR products isdetected following their separation and accurate sizing that is achievedby slab or capillary electrophoresis. In one embodiment, capillaryelectrophoresis is used, and it can be performed in microfabricatedchannels or capillary arrays. Alternatively, methods utilizing massspectrometry and microarray technology are used. Multiplex STR analysiscan be performed to determine fetal fraction usingcommercially-available kits e.g. AmpFλSTR® Identifier® PCR AmplificationKit (FIG. 4) and AmpFλSTR® MiniFiler® PCR Amplification Kit (FIG. 5)(Applied Biosystems, Foster City, Calif.). The AmpFλSTR® MiniFiler® PCRAmplification Kit was designed to amplify as miniSTRs eight of thelargest sized loci in the AmpFλSTR® Identifier® PCR Amplification Kit.Together with the gender-identification locus Amelogenin, the nine-locusmultiplex enables simultaneous amplification of loci of cfDNA samples(see Example 10).

In one embodiment, multiplex STR analysis for determining fetal fractionis performed by amplifying polymorphic target nucleic acids present in amaternal plasma sample that each comprise a miniSTR selected fromCSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317,D16S539, D18S51, D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63, D11S4463,D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500,D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627,and D1GATA113. In another embodiment, multiplex STR analysis fordetermining fetal fraction is performed by amplifying polymorphic targetnucleic acids present in a maternal plasma sample for the panel ofminiSTRs: CSF1PO, D13S317, D16 S539, D18S51, D21S11, D2S1338, D7S820 andFGA. The miniSTRs can be located on the same or on differentchromosomes. The method is a fetal gender-independent method. Therefore,in some embodiments, the miniSTRs are located on chromosomes other thanthe Y chromosome. In other embodiments, the miniSTRs are located onchromosomes other than chromosomes 13, 18, 21 or X i.e. chromosomes thatmight be involved in an aneuploidy.

Samples of maternal plasma often contain less than 100 pg of cfDNA. Thelow copy number DNA samples can fall below the sensitivity limitationsof STR analysis methods. The intractable samples can be made amenable byincreasing the number of starting cIDNA available for subsequent STRanalysis by a whole genome amplification strategy. In one embodiment,the mixture of fetal and maternal nucleic acids can be preamplifiedbefore alleles are detected or quantified. For example, templatecell-free DNA can be amplified by PCR. The nucleic acid can be amplifiedfor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 cycles. Nucleic acid can be amplified for about 1-10cycles, about 1-20 cycles, about 1-30 cycles, about 1-40 cycles, about5-15 cycles, about 5-20 cycles, about 5-30 cycles, about 5-40 cycles,about 10-15 cycles, about 10-20 cycles, about 10-30 cycles, about 10-40cycles, about 20-30 cycles, about 20-40 cycles, or about 30-40 cycles.The amount of template nucleic acid that can be amplified can about10-1000 pg, 25-1000 pg, 50-1000 pg, 100-1000, pg, 200-1000 pg, 300-1000pg, 400-1000 pg, 500-1000 pg, 600-1000, pg, 700-1000 pg, or 800-1000 pg.Following preamplification, the nucleic acids can be diluted beforealleles are detected or quantified. Preamplification can be used toincrease the detection sensitivity of alleles in a sample, for example,a maternal sample (Example 11). In another embodiment, genotyping apolymorphism need not require a pre-amplification step. Any PCR-basedamplification method can be used to preamplify the cIDNA. Amplificationmethods include but are not limited to whole genome amplificationstrategies including methods such as primer extension preamplification,degenerate oligonucleotide-primed PCR (DOP-PCR), low fragments from lowquantities of DOP-PCR, improved primer extension preamplification PCR(IPEP PCR), and modified improved primer extension preamplification(mIPEP). Thus, in one embodiment, the method that is used fordetermining the fetal fraction in a maternal sample e.g. plasma sample,comprises preamplifying the mixture of fetal and maternal nucleic acidspresent in the plasma cIDNA sample using a whole genome amplificationmethod, amplifying a plurality of polymorphic nucleic acids in saidmixture of fetal and maternal nucleic acids, wherein each of said atleast one polymorphic nucleic acid comprises an STR; determining theamount of fetal and maternal STR alleles at least one polymorphicnucleic acid; and calculating the fetal fraction from the amount offetal and maternal STR alleles. Following preamplification, multiplexSTR analysis for determining fetal fraction is performed by amplifyingpolymorphic target nucleic acids present in a maternal plasma samplethat each comprise a miniSTR selected from CSF1PO, FGA, TH01, vWA,D3S1358, D5S818, D7S820, D8S1179, D13S317, D16 S539, D18S51, D2S1338,Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301,D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157,D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529,D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.Alternatively, multiplex STR analysis for determining fetal fraction isperformed by amplifying polymorphic target nucleic acids for the panelof miniSTRs: CSF1PO, D13S317, D16 S539, D18S51, D21S11, D2S1338, D7S820and FGA.

Applications

Methods described herein are applicable to diagnosis or prognosis ofvarious disease conditions including, but not limited to, cancer,genetic disorders and infection. The fetal fraction of nucleic acid in amaternal sample can be used for determining a chromosomal abnormality.Examples of chromosomal abnormalities include, for example, aneuploidy,monosomy, trisomy, duplication, inversion, deletion, polyploidy,deletion of a part of a chromosome, addition, addition of a part ofchromosome, insertion, a fragment of a chromosome, a region of achromosome, chromosomal rearrangement, and translocation. For example,aneuploidy can refer to the occurrence of one or more extra or missingchromosomes in a sample.

Examples of fetal conditions that can be determined using the methods ofthe provided invention include, for example, Angleman syndrome(15q11.2-q13), cri-du-chat syndrome (5p-), DiGeorge syndrome andVelo-cardiofacial syndrome (22q11.2), Miller-Dieker syndrome (17 p13.3),Prader-Willi syndrome (15q11.2-q13), retinoblastoma (13q14),Smith-Magenis syndrome (17 p11.2), trisomy 13, trisomy 16, trisomy 18,trisomy 21 (Down's syndrome), triploidy, Williams syndrome (7q 11.23),and Wolf-Hirschhom syndrome (4p-). Examples of sex chromosomeabnormalities that can be detected by methods described herein include,but are not limited to, Kallman syndrome (Xp22.3), steroid sulfatedeficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22.3), Klinefeltersyndrome (XXY), fragile X syndrome, Turner syndrome metafemales ortrisomy X, and monosomy X.

In one embodiment, fetal fraction information can be used to setthresholds and estimate minimum sample size in aneuploidy detection.Such use is described in Example 7 below. Fetal fraction information canbe used in conjunction with sequencing information. For example, nucleicacids from a cell-free sample, for example a maternal plasma or serumsample, can be used to enumerate sequences in a sample. Sequences can beenumerated using any of the sequencing techniques described above.Knowledge of fetal fraction can be used to set “cutoff” thresholds tocall “aneuploidy,” “normal,” or “marginal/no call” (uncertain) states.Then, calculations can be performed to estimate the minimum number ofsequences required to achieve adequate sensitivity (i.e. probability ofcorrectly identifying an aneuploidy state).

The determination of fetal fraction according to the method of theinvention can be practiced in combination with any method used todetermine the presence of absence of fetal aneuploidy in a maternalplasma sample. In addition to the method described herein for thedetermination of aneuploidy, the determination of fetal fraction bymassively parallel sequencing can be used in conjunction with othermethods for determining fetal aneuploidy, for example, according to themethods described in U.S. U.S. Patent Application Publication Nos. US2007/0202525A1; US2010/0112575A1, US 2009/0087847A1; US2009/0029377A1;US 2008/0220422A1; US2008/0138809A1, US2008/0153090A1, and U.S. Pat. No.7,645,576. The methods can also be combined with assays for determiningother prenatal conditions associated with the mother and/or the fetus.For example, the method can be used in conjunction with prenatalanalyses, for example, as described in U.S. Patent ApplicationPublication Nos. US2010/0112590A1, US2009/0162842A1, US2007/0207466A1,and US2001/0051341A1, all of which are incorporated by reference intheir entirety.

The methods described can be applied to determine the fraction of anyone population of nucleic acids in a mixture of nucleic acidscontributed by different genomes. In addition to determining thefraction contributed to a sample by two individuals e.g. the differentgenomes are contributed by the fetus and the mother carrying the fetus,the methods can be used to determine the fraction of a genome in amixture derived from two different cells of from one individual e.g. thegenomes are contributed to the sample by aneuploid cancerous cells andnormal euploid cells from the same subject.

Compositions and Kits

The present invention is also directed to compositions and kits orreagent systems useful for practicing the methods described herein.

The compositions of the invention can be included in kits for massivelyparallel sequencing mixtures of fetal and maternal nucleic acidmolecules e.g. cfDNA, present in a maternal sample e.g. a plasma sample.The kits comprise a composition comprising at least one set of primersfor amplifying at least one polymorphic target nucleic acid in saidfetal and maternal nucleic acid molecules. Polymorphic target nucleicacids can comprise without limitation single nucleotide polymorphisms(SNPs), tandem SNPs, small-scale multi-base deletions or insertions,called IN-DELS (also called deletion insertion polymorphisms or DIPs),Multi-Nucleotide Polymorphisms (MNPs) Short Tandem Repeats (STRs),restriction fragment length polymorphism (RFLP), or a polymorphismcomprising any other change of sequence in a chromosome. Sequencingmethods utilizing the compositions of the invention are NGS methods ofsingle nucleic acid molecules or clonally amplified nucleic acidmolecules as described herein. The massively parallel sequencing methodsof NGS include pyrosequencing, sequencing by synthesis with reversibledye terminators, real-time sequencing, or sequencing by oligonucleotideprobe ligation.

In one embodiment, the compositions includes primers for amplifyingpolymorphic target nucleic acids that each comprise at least one SNP. Inone embodiment, the at least one SNP is selected from SNPs rs560681 (SEQID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 &6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10),rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14),rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18),rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261(SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ IDNOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 &32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36),rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696(SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ IDNOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 &50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), andrs530022 (SEQ ID NOS 55 & 56). Exemplary corresponding sets of primersfor amplifying the SNPs are provided as SEQ ID NOs:57-112.

In another embodiment, the composition includes primers for amplifyingpolymorphic target nucleic acids that each comprise at least one tandemSNP. In one embodiment, the composition includes primers for amplifyingtandem SNPs. In one embodiment, the composition includes primers foramplifying the tandem SNPs disclosed herein, and the compositioncomprises the corresponding exemplary primers of SEQ ID NOS:197-310.

In another embodiment, the composition includes primers for amplifyingpolymorphic target nucleic acids that each comprise at least one STR.Exemplary STRs include CSF1PO, FGA, TH01, TPDX, vWA, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16 S539, D18S51, D21S11, D2S1338, Penta D,Penta E, D22S1045, D20 S1082, D20 S482, D18S853, D17S1301, D175974,D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122,D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053,D2S1776, D2S441, D1S1677, D1S1627 and D1GATA113, which can be amplifiedby the corresponding sets of primers of SEQ ID NOs:113-196.

Kits can contain a reagent combination including the elements requiredto conduct an assay according to the methods disclosed herein. Thereagent system is presented in a commercially packaged form, as acomposition or admixture where the compatibility of the reagents willallow, in a test device configuration, or more typically as a test kit,i.e., a packaged combination of one or more containers, devices, or thelike holding the necessary reagents, and preferably including writteninstructions for the performance of assays. The kit of the presentinvention may be adapted for any configuration of assay and may includecompositions for performing any of the various assay formats describedherein. Kits for determining fetal fraction comprise compositionsincluding primer sets for amplifying polymorphic nucleic acids presentin a maternal sample as described and, where applicable, reagents forpurifying cIDNA, are within the scope of the invention. In oneembodiment, a kit designed to allow quantification of fetal and maternalpolymorphic sequences e.g. STRs and/or SNPs and/or tandem SNPs, in acfDNA plasma sample, includes at least one set of allele specificoligonucleotides specific for a selected SNP and/or region of tandemrepeats. Preferably, the kit includes a plurality of primer sets toamplify a panel of polymorphic sequences. A kit can comprise otherreagents and/or information for genotyping or quantifying alleles in asample (e.g., buffers, nucleotides, instructions). The kits also includea plurality of containers of appropriate buffers and reagents.

The present invention is described in further detail in the followingExamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.The following examples are offered to illustrate, but not to limit theclaimed invention.

EXPERIMENTAL Example 1 Determination of Fetal Fraction using MassivelyParallel Sequencing: Sample Processing and cfDNA Extraction

Peripheral blood samples were collected from pregnant women in theirfirst or second trimester of pregnancy and who were deemed at risk forfetal aneuploidy. Informed consent was obtained from each participantprior to the blood draw. Blood was collected before amniocentesis orchorionic villus sampling. Karyotype analysis was performed using thechorionic villus or amniocentesis samples to confirm fetal karyotype.

Peripheral blood drawn from each subject was collected in ACD tubes. Onetube of blood sample (approximately 6-9 mL/tube) was transferred intoone 15-mL low speed centrifuge tube. Blood was centrifuged at 2640 rpm,4° C. for 10 min using Beckman Allegra 6 R centrifuge and rotor model GA3.8.

For cell-free plasma extraction, the upper plasma layer was transferredto a 15-ml high speed centrifuge tube and centrifuged at 16000×g, 4° C.for 10 min using Beckman Coulter Avanti J-E centrifuge, and JA-14 rotor.The two centrifugation steps were performed within 72 h after bloodcollection. Cell-free plasma comprising cfDNA was stored at −80° C. andthawed only once before amplification of plasma cIDNA or forpurification of cIDNA.

Purified cell-free DNA (cfDNA) was extracted from cell-free plasma usingthe QIAamp Blood DNA Mini kit (Qiagen) essentially according to themanufacturer's instruction. One milliliter of buffer AL and 100 μl ofProtease solution were added to 1 ml of plasma. The mixture wasincubated for 15 minutes at 56° C. One milliliter of 100% ethanol wasadded to the plasma digest. The resulting mixture was transferred toQIAamp mini columns that were assembled with VacValves and VacConnectorsprovided in the QIAvac 24 Plus column assembly (Qiagen). Vacuum wasapplied to the samples, and the cIDNA retained on the column filters waswashed under vacuum with 750 μl of buffer AW1, followed by a second washwith 750 μl of buffer AW24. The column was centrifuged at 14,000 RPM for5 minutes to remove any residual buffer from the filter. The cfDNA waseluted with buffer AE by centrifugation at 14,000 RPM, and theconcentration determined using Qubit™ Quantitation Platform(Invitrogen).

Example 2 Determination of Fetal Fraction using Massively ParallelSequencing: Preparation of Sequencing Libraries, Sequencing, andAnalysis of Sequencing Data

a. Preparation of Sequencing Libraries

All sequencing libraries i.e. target, primary and enriched libraries,were prepared from approximately 2 ng of purified cIDNA that wasextracted from maternal plasma. Library preparation was performed usingreagents of the NEBNext™ DNA Sample Prep DNA Reagent Set 1 (Part No.E6000L; New England Biolabs, Ipswich, Mass.) for Illumina® as follows.Because cell-free plasma DNA is fragmented in nature, no furtherfragmentation by nebulization or sonication was done on the plasma DNAsamples. The overhangs of approximately 2 ng purified cfDNA fragmentscontained in 40 μl were converted into phosphorylated blunt endsaccording to the NEBNext® End Repair Module by incubating in a 1.5 mlmicrofuge tube the cIDNA with 5 μl 10× phosphorylation buffer, 2 μldeoxynucleotide solution mix (10 mM each dNTP), 1 μl of a 1:5 dilutionof DNA Polymerase I, 1 μl T4 DNA Polymerase and 1 μl T4 PolynucleotideKinase provided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1 for 15minutes at 20° C. The enzymes were then heat inactivated by incubatingthe reaction mixture at 75° C. for 5 minutes. The mixture was cooled to4° C., and dA tailing of the blunt-ended DNA was accomplished using 10μl of the dA-tailing master mix containing the Klenow fragment (3′ to 5′exo minus) (NEBNext™ DNA Sample Prep DNA Reagent Set 1), and incubatingfor 15 minutes at 37° C. Subsequently, the Klenow fragment was heatinactivated by incubating the reaction mixture at 75° C. for 5 minutes.Following the inactivation of the Klenow fragment, 1 μl of a 1:5dilution of Illumina Genomic Adaptor Oligo Mix (Part No. 1000521;Illumina Inc., Hayward, Calif.) was used to ligate the Illumina adaptors(Non-Index Y-Adaptors) to the dA-tailed DNA using 4 μl of the T4 DNAligase provided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1, byincubating the reaction mixture for 15 minutes at 25° C. The mixture wascooled to 4° C., and the adaptor-ligated cIDNA was purified fromunligated adaptors, adaptor dimers, and other reagents using magneticbeads provided in the Agencourt AMPure XP PCR purification system (PartNo. A63881; Beckman Coulter Genomics, Danvers, Mass.). Eighteen cyclesof PCR were performed to selectively enrich adaptor-ligated cIDNA usingPhusion® High-Fidelity Master Mix (Finnzymes, Woburn, Mass.) andIllumina's PCR primers complementary to the adaptors (Part No. 1000537and 1000537). The adaptor-ligated DNA was subjected to PCR (98° C. for30 seconds; 18 cycles of 98° C. for 10 seconds, 65° C. for 30 seconds,and 72° C. for 30 seconds; final extension at 72° C. for 5 minutes, andhold at 4° C.) using Illumina Genomic PCR Primers (Part Nos. 100537 and1000538) and the Phusion HF PCR Master Mix provided in the NEBNext™ DNASample Prep DNA Reagent Set 1, according to the manufacturer'sinstructions. The amplified product was purified using the AgencourtAMPure XP PCR purification system (Agencourt Bioscience Corporation,Beverly, Mass.) according to the manufacturer's instructions availableat www.beckmangenomics.com/products/AMPureXPProtocol_(—)000387v001.pdf.The purified amplified product was eluted in 40 μl of Qiagen EB Buffer,and the concentration and size distribution of the amplified librarieswas analyzed using the Agilent DNA 1000 Kit for the 2100 Bioanalyzer(Agilent technologies Inc., Santa Clara, Calif.).

b. Sequencing

Sequencing of library DNA was performed using the Genome Analyzer II(Illumina Inc., San Diego, Calif., USA) according to standardmanufacturer protocols. Copies of the protocol for whole genomesequencing using Illumina/Solexa technology may be found atBioTechniques® Protocol Guide 2007 Published December 2006: p 29, and onthe world wide web atbiotechniques.com/default.asp?page=protocol&subsection=article_display&id=112378.The DNA library was diluted to 1 nM and denatured. Library DNA (5 pM)was subjected to cluster amplification according to the proceduredescribed in Illumina's Cluster Station User Guide and Cluster StationOperations Guide, available on the world wide web atillumina.com/systems/genome analyzer/cluster_station.ilmn. The amplifiedDNA was sequenced using Illumina's Genome Analyzer II to obtainsingle-end reads of 36 bp. Only about 30 bp of random sequenceinformation are needed to identify a sequence as belonging to a specifichuman chromosome. Longer sequences can uniquely identify more particulartargets. In the present case, a large number of 36 by reads wereobtained, covering approximately 10% of the genome.

c. Analysis of Sequencing Data for the Determination of Fetal Fraction

Upon completion of sequencing of the sample, the Illumina “SequencerControl Software” transferred image and base call files to a Unix serverrunning the Illumina “Genome Analyzer Pipeline” software version 1.51.the 36 bp reads were aligned to an artificial reference genome e.g. aSNP genome, using the BOWTIE program. The artificial reference genomewas identified as the grouping of the polymorphic DNA sequences thatencompass the alleles comprised in the polymorphic target sequences. Forexample, the artificial reference genome is a SNP genome comprising SEQID NOs: 1-56. Only reads that mapped uniquely to the artificial genomewere used for the analysis of fetal fraction. Reads that matchedperfectly to the SNP genome were counted as tags and filtered. Of theremaining reads, only reads having one or two mismatches were counted astags and included in the analysis. Tags mapped to each of thepolymorphic alleles were counted, and the fetal fraction was determinedas a percent of the ratio of the number of tags mapped to the majorallele i.e. maternal allele, and the number of tags mapped to the minorallele i.e. fetal allele.

Example 3

Selection of Autosomal SNPs for the Determination of Fetal Fraction

A set of 28 autosomal SNPs were selected from a list of 92 SNPs (Pakstiset al., Hum Genet 127:315-324 [2010]) and from Applied Biosystems byLife Technologies® (Carlsbad, Calif.) at world wide web addressappliedbiosystems.com, and validated for use in multiplexed PCRamplification. Primers were designed to hybridize to a sequence close tothe SNPs site on the cfDNA to ensure that it be included in the 36 byread generated from the massively parallel sequencing on the IlluminaAnalyzer GII, and to generate amplicons of sufficient length to undergobridge-amplification during cluster formation. Thus, primers weredesigned to generate amplicons that were at least 110 bp, which whencombined with the universal adaptors (Illumina Inc., San Diego, Calif.)used for cluster amplification, resulted in DNA molecules of at least200 bp. Primer sequences were identified, and primer sets i.e. forwardand reverse primers, were synthesized by Integrated DNA Technologies(San Diego, Calif.), and stored as a 1 μM solution to be used foramplifying polymorphic target sequences as described in Examples 4-7.Table 1 provides the RefSNP (rs) accession ID numbers, the primers usedfor amplifying the target cIDNA sequence, and the sequences of theamplicons comprising the possible SNP alleles that would be generatedusing the primers. The SNPs given in Table 1 were used for thesimultaneous amplification of 13 target sequences in a multiplexedassay. The panel provided in Table 1 is an exemplary SNP panel. Fewer ormore SNPs can be employed to enrich the fetal and maternal DNA forpolymorphic target nucleic acids. Additional SNPs that can be usedinclude the SNPs given in Table 2. The SNP alleles are shown in bold andare underlined. Other additional SNPs that can be used to determinefetal fraction according to the present method include rs315791,rs3780962, rs1410059, rs279844, rs38882, rs9951171 (SEQ ID NOS 29 & 30),rs214955, rs6444724, rs2503107, rs1019029, rs1413212, rs1031825,rs891700, rs1005533, rs2831700, rs354439, rs1979255, rs1454361,rs8037429, and rs1490413, which have been analyzed for determining fetalfraction by TaqMan PCR, and are disclosed in U.S. Provisionalapplications 61/296,358 and 61/360,837.

TABLE 1 SNP Panel for the Determination of Fetal Fraction Forward PrimerSequence, Reverse Primer Amplicon: Amplicon: name and Sequence, nameSNP ID Chr Allele 1 Allele 2 SEQ ID NO: and SEQ ID NO: rs560681 1CACATGCACAGCCA CACATGCACAGCCA CACATGCA CCCCAAGGTCC GCAACCCTGTCAGCGCAACCCTGTCAGC CAGCCAGC TGTGACCTGAG AGGAGTTCCCACCA AGGAGTTCCCACCA AACCCT GTTTCTTTCTGAGAA GTTTCTTTCTGAGAA (rs560681_C1_ (rs560681_C1_1_R;CATCTGTTCAGGTTT CATCTGTTCAGGTTT 1_F; SEQ ID SEQ ID NO: 58) CTCTCCATCTCTA TT CTCTCCATCTCT G TT NO: 57) TACTCAGGTCACAG TACTCAGGTCACAGGACCTTGGGG (SEQ GACCTTGGGG (SEQ ID NO: 1) ID NO: 2) rs1109037 2TGAGGAAGTGAGGC TGAGGAAGTGAGGC TGAGGAAG TGCCAGTGCGA TCAGAGGGTAAGAATCAGAGGGTAAGAA TGAGGCTC GATGAAAGTCT ACTTTGTCACAGAGC ACTTTGTCACAGAGCAGAGGGT TT TGGTGGTGAGGGTG TGGTGGTGAGGGTG (rs110937_C2_ (rs110937_C2_1_R;GAGATTTTACACTCC GAGATTTTACACTCC 1_F; SEQ ID SEQ ID NO: 60)CTGCCTCCCACACCA CTGCCTCCCACACCA NO: 59) GTTTCTCC A GAGTGG GTTTCTCC GGAGTGG AAAGACTTTCATCTC AAAGACTTTCATCTC GCACTGGCA (SEQ IDGCACTGGCA (SEQ ID NO: 3) NO: 4) rs9866013 3 GTGCCTTCAGAACCTGTGCCTTCAGAACCT GTGCCTTCA TCCCATCCCAC TTGAGATCTGATTCT TTGAGATCTGATTCTGAACCTTTG CAGCCACCC ATTTTTAAAGCTTCT ATTTTTAAAGCTTCT AGATCTGAT(rs9866013_C3_1_ TAGAAGAGAGATTG TAGAAGAGAGATTG (rs9866013_C3_R; SEQ ID NO: 62) CAAAGTGGGTTGTTT CAAAGTGGGTTGTTT 1_F; SEQCTCTAGCCAGACAG CTCTAGCCAGACAG ID NO: 61) GGCAGG C AAATAGG GGCAGG TAAATAGG GGTGGCTGGTGGGA GGTGGCTGGTGGGA TGGGA (SEQ ID NO: 5)TGGGA (SEQ ID NO: 6) rs13182883 5 AGGTGTGTCTCTCTT AGGTGTGTCTCTCTTAGGTGTGTC CCTTTGTCCCAC TTGTGAGGGGAGGG TTGTGAGGGGAGGG TCTCTTTTG CTCCCCACCGTCCCTTCTGGCCTA GTCCCTTCTGGCCTA TGAGGGG (rs13182883_C5_ GTAGAGGGCCTGGCGTAGAGGGCCTGGC (rs13182883_ 1_R; SEQ ID CTGCAGTGAGCATTC CTGCAGTGAGCATTCC5_1_F; SEQ NO: 64) AAATCCTC A AGGAA AAATCCTC G AGGAA ID NO: 63)CAGGGTGGGGAGGT CAGGGTGGGGAGGT GGGACAAAGG (SEQ GGGACAAAGG (SEQ ID NO: 7)ID NO: 8) rs13218440 6 CCTCGCCTACTGTGC CCTCGCCTACTGTGC CCTCGCCTACCATCCCAGCT TGTTTCTAACCATCA TGTTTCTAACCATCA CTGTGCTGT GAGTATTCCAGTGCTTTTCCCTGAAT TGCTTTTCCCTGAAT TTCTAACC GAG CTCTTGAGTCTTTTTCTCTTGAGTCTTTTT (rs13218440_ (rs13218440_C6_ CTGCTGTGGACTGACTGCTGTGGACTGA C6_1_F; SEQ 1_R; SEQ ID AACTTGATCCTGAG AACTTGATCCTGAGID NO: 65) NO: 66) ATTCACCTCTAGTCC ATTCACCTCTAGTCC CTCTG A GCAGCCTCCCTCTG G GCAGCCTCC TGGAATACTCAGCT TGGAATACTCAGCT GGGATGG (SEQ IDGGGATGG (SEQ ID NO: 9) NO: 10) rs7041158 9 AATTGCAATGGTGA AATTGCAATGGTGAAATTGCAAT CCAGTGAGAAG GAGGTTGATGGTAA GAGGTTGATGGTAA GGTGAGAG TGTCTTGGGTTAATCAAACGGAACT AATCAAACGGAACT GTTGATGGT GG (SEQ ID TGTTATTTTGTCATTTGTTATTTTGTCATT (SEQ ID NO: 68) CTGATGGACTGGAA CTGATGGACTGGAA NO: 67)CTGAGGATTTTCAAT CTGAGGATTTTCAAT TTCCT C TCCAACCCA TTCCT T TCCAACCCAAGACACTTCTCACTG AGACACTTCTCACTG G (SEQ ID NO: 11) G (SEQ ID NO: 12)rs740598 10 GAAATGCCTTCTCAG GAAATGCCTTCTCAG GAAATGCC GGTTTGAGCAGGTAATGGAAGGTTA GTAATGGAAGGTTA TTCTCAGGT TTCTGAGAATG TCCAAATATTTTTCGTCCAAATATTTTTCG AATGGAAG TGGCT (SEQ ID TAAGTATTTCAAATA TAAGTATTTCAAATAGT (SEQ ID NO: 70) GCAATGGCTCGTCTA GCAATGGCTCGTCTA NO: 69) TGGTTAGTCTC ACAG TGGTTAGTCTC G CAG CCACATTCTCAGAAC CCACATTCTCAGAAC TGCTCAAACC (SEQTGCTCAAACC (SEQ ID NO: 13) ID NO: 14) rs10773760 12 ACCCAAAACACTGGACCCAAAACACTGG ACCCAAAA CCCTTATCTGCT AGGGGCCTCTTCTCA AGGGGCCTCTTCTCACACTGGAG ATGTGGCATAC TTTTCGGTAGACTGC TTTTCGGTAGACTGC GGGCCT TTGG (SEQ IDAAGTGTTAGCCGTC AAGTGTTAGCCGTC (SEQ ID NO: 72) GGGACCAGCTTCTGTGGGACCAGCTTCTGT NO: 71) CTGGAAGTTCGTCA CTGGAAGTTCGTCA AATTGCAGTTA A GTCAATTGCAGTTA G GT CAAGTATGCCACAT CCAAGTATGCCACA AGCAGATAAGGGTAGCAGATAAGGG (SEQ ID NO: 15) (SEQ ID NO: 16) rs4530059 14GCACCAGAATTTAA GCACCAGAATTTAA GCACCAGA GCACCTGACAG ACAACGCTGACAATACAACGCTGACAAT ATTTAAACA GCACATCAGCG AAATATGCAGTCGA AAATATGCAGTCGAACGCTGAC (SEQ ID NO: 74) TGATGACTTCCCAGA TGATGACTTCCCAGA AA (SEQ IDGCTCCAGAAGCAAC GCTCCAGAAGCAAC NO: 73) TCCAGCACAC A GAG TCCAGCACAC G GAGAGGCGCTGATGTGC AGGCGCTGATGTGC CTGTCAGGTGC (SEQ CTGTCAGGTGC (SEQID NO: 17) ID NO: 18) rs7205345 16 TGACTGTATACCCCA TGACTGTATACCCCATGACTGTAT GCACTAAGGAT GGTGCACCCTTGGGT GGTGCACCCTTGGGT ACCCCAGGGTGGAAGTCTA CATCTCTATCATAGA CATCTCTATCATAGA TGCACCC GTGTG (SEQ IDACTTATCTCACAGAG ACTTATCTCACAGAG (SEQ ID NO: 76) TATAAGAGCTGATTTTATAAGAGCTGATTT NO: 75) CTGTGTCTGCCT C TC CTGTGTCTGCCT G TCACACTAGACTTCCAC ACACTAGACTTCCAC ATCCTTAGTGC (SEQ ATCCTTAGTGC (SEQID NO: 19) ID NO: 20) rs8078417 17 TGTACGTGGTCACCA TGTACGTGGTCACCATGTACGTGG AGTGTGAGAAG GGGGACGCCTGGCG GGGGACGCCTGGCG TCACCAGG AGCCTCAAGGACTGCGAGGGAGGCC CTGCGAGGGAGGCC GGACG (SEQ CAGC (SEQ ID CCGAGCCTCGTGCCCCCGAGCCTCGTGCCC ID NO: 77) NO: 78) CCGTGAAGCTTCAG CCGTGAAGCTTCAGCTCCCCTCCC C GGCT CTCCCCTCCC T GGCT GTCCTTGAGGCTCTT GTCCTTGAGGCTCTTCTCACACT (SEQ ID CTCACACT (SEQ ID NO: 21) NO: 22) rs576261 19CAGTGGACCCTGCT CAGTGGACCCTGCT CAGTGGAC GTGGCAAAGGA GCACCTTTCCTCCCCGCACCTTTCCTCCCC CCTGCTGCA GAGAGTTGTGA TCCCATCAACCTCTT TCCCATCAACCTCTTCCTT (SEQ GG (SEQ ID TTGTGCCTCCCCCTC TTGTGCCTCCCCCTC ID NO: 79) NO: 80)CGTGTACCACCTTCT CGTGTACCACCTTCT CTGTCACCA A CCCTG CTGTCACCA C CCCTGGCCTCACAACTCTCT GCCTCACAACTCTCT CCTTTGCCAC (SEQ CCTTTGCCAC (SEQID NO: 23) ID NO: 24) rs2567608 20 CAGTGGCATAGTAG CAGTGGCATAGTAGCAGTGGCA CCTCTCCGACA TCCAGGGGCTCCTCC TCCAGGGGCTCCTCC TAGTAGTCCACTTCCGCCG TCAGCACCTCCAGC TCAGCACCTCCAGC AGGGGCT (SEQ ID NO: 82)ACCTTCCAGGAGGC ACCTTCCAGGAGGC (SEQ ID AGCAGCGCAGGCAG AGCAGCGCAGGCAGNO: 81) AGAACCCGCTGGAA AGAACCCGCTGGAA G A ATCGGCGGAAGT G G ATCGGCGGAAGTTGTCGGAGAGG (SEQ TGTCGGAGAGG (SEQ ID NO: 25) ID NO: 26)

TABLE 2 Additional SNPs for the Determination of Fetal Fraction ForwardPrimer Sequence, Reverse Primer Amplicon: Amplicon: name andSequence, name SNP ID Chr Allele 1 Allele 2 SEQ ID NO: and SEQ ID NO:rs430046 16 AGGTCTGGGGGCC AGGTCTGGGGGCCGC AGGTCTGG TCCTCCCATTAGCTGAATGCCAAGC TGAATGCCAAGCTGG GGGCCGCT AACCCAGCACC TGGGAATCTTAAATGAATCTTAAATGTTA GAAT T GTTAAGGAACAAG AGGAACAAGGTCATA (rs430046_C1_(rs430046_C1_1_R GTCATACAATGAAT CAATGAATGGTGTGA 1_F; SEQ ID ;SEQ ID NO: 84) GGTGTGATGTAAAA TGTAAAAGCTTGGGA NO: 83) GCTTGGGAGGTGATGGTGATTT T TGAGGG TT C TGAGGGTAGGT TAGGTGCTGGGTTTA GCTGGGTTTAATGGATGGGAGGA (SEQ ID GAGGA (SEQ ID NO: 28) NO: 27) rs9951171 18ACGGTTCTGTCCTG ACGGTTCTGTCCTGT ACGGTTCTG CCTGTTCACTTG TAGGGGAGAAAAGAGGGGAGAAAAGTCC TCCTGTAGG TGGCAGGGCA TCCTCGTTGTTCCT TCGTTGTTCCTCTGGGGGAGA (rs9951171_C1_1_ CTGGGATGCAACAT ATGCAACATGAGAGA (rs9951171_C1_R; SEQ ID NO: 86) GAGAGAGCAGCAC GCAGCACACTGAGGC 1_F; SEQ ACTGAGGCTTTATGTTTATGG G TTGCCCT ID NO: 85) G A TTGCCCTGCCAC GCCACAAGTGAACAGAAGTGAACAGG G (SEQ ID NO: 30) (SEQ ID NO: 29) rs338882 5 GCGCAGTCAGATGGCGCAGTCAGATGGG GCGCAGTC TCCAGCCCTTG GGCGTGCTGGCGTC CGTGCTGGCGTCTGTAGATGGGC TCCCAAACGTG TGTCTTCTCTCTCTC CTTCTCTCTCTCCTGC GTGC TCTGCTCTCTGGCTT TCTCTGGCTTCATTTT (rs338882_C1_ (rs338882_C1_1_R;CATTTTTCTCTCCTT TCTCTCCTTCTGTCTC 1_F; SEQ ID SEQ ID NO: 88)CTGTCTCACCTTCT ACCTTCTTTCGTGTGC NO: 87) TTCGTGTGCCTGTG CTGTGCA T ACACACGCA C ACACACGTTTG TTTGGGACAAGGG GGACAAGGG CTGGA (SEQ ID NO: 32)CTGGA (SEQ ID NO: 31) rs10776839 9 GCCGGACCTGCGA GCCGGACCTGCGAAAGCCGGACC CGGGCAACTGG AATCCCAAAATGCC TCCCAAAATGCCAAA TGCGAAAT GGCTCTGATCAAACATTCCCGCCT CATTCCCGCCTCACA CCCAA (rs10776839_C1_1_ CACATGATCCCAGATGATCCCAGAGAGAG (rs10776839C1_ R; SEQ ID GAGAGGGGACCCA GGGACCCAGTGTTCC1_F; SEQ NO: 90) GTGTTCCCAGCTTG CAGCTTGCAGCTGAG ID NO: 89) CAGCTGAGGAGCCGAGCCCGAG T TTGCC CGAG G TTGCCGTCA GTCAGATCAGAGCCC GATCAGAGCCCCACAGTTGCCCG (SEQ ID GTTGCCCG (SEQ ID NO: 34) NO: 33) rs9905977 17AGCAGCCTCCCTCG AGCAGCCTCCCTCGA AGCAGCCT GGCAGAGGGGA ACTAGCTCACACTACTAGCTCACACTACG CCCTCGACT AAGACGAAAGG CGATAAGGAAAAT ATAAGGAAAATTCAT AGCTA TCATGAGCTGGTGT GAGCTGGTGTCCAAG (rs9905977_C1_ (rs9905977_C1_1_CCAAGGAGGGCTG GAGGGCTGGGTGACT 1_F; SEQ R; SEQ ID NO: 92) GGTGACTCGTGGCTCGTGGCTCAGTCAGC ID NO: 91) CAGTCAGC A TCAAG G TCAAGATTCCTTTCATTCCTTTCGTCTTT GTCTTTCCCCTCTGCC CCCCTCTGCC (SEQ (SEQ ID NO: 36)ID NO: 35) rs1277284 4 TGGCATTGCCTGTA TGGCATTGCCTGTAA TGGCATTGCAAGCACCATTC ATATACATAGCCAT TATACATAGCCATGG CTGTAATAT TAATGATTTTGGGTTTTTTATAGGC TTTTTTATAGGCAATT ACATAG G AATTTAAGATGAAT TAAGATGAATAGCTT(rs1277284_C4_ (rs1277284_C4_1_ AGCTTCTAAACTAT CTAAACTATAGATAA 1_F; SEQR; SEQ ID NO: 94) AGATAAGTTTCATT GTTTCATTACCCCAG ID NO: 93)ACCCCAGGAAGCT GAAGCTGAACTATAG GAACTATAGCTACT CTACTTT C CCCAAAA TT ACCCAAAATCAT TCATTAGAATGGTGC TAGAATGGTGCTT TT (SEQ ID NO: 38)(SEQ ID NO: 37) rs258684 7 ATGAAGCCTTCCAC ATGAAGCCTTCCACC ATGAAGCCGATCAGTTGTT CAACTGCCTGTATG AACTGCCTGTATGAC TTCCACCAA GTTTCTATATTTACTCATCTGGGGAC TCATCTGGGGACTTC CTG CCTT TTCTGCTCTATACT TGCTCTATACTCAAA(rs258684_C7_ (rs258684_C7_1_R; CAAAGTGGCTTAGT GTGGCTTAGTCACTG1_F; SEQ ID SEQ ID NO: 96) CACTGCCAATGTAT CCAATGTATTTCCAT NO: 95)TTCCATATGAGGGA ATGAGGGACG G TGAT CG A TGATTACTAAG TACTAAGGAAATATAGAAATATAGAAAC GAAACAACAACTGAT AACAACTGATC C (SEQ ID NO: 40)(SEQ ID NO: 39) rs1347696 8 ACAACAGAATCAG ACAACAGAATCAGGT ACAACAGACTGAACTGAAC GTGATTGGAGAAA GATTGGAGAAAAGAT ATCAGGTG AAAGAATTAAGAGATCACAGGCCTA CACAGGCCTAGGCAC ATTGGA GTC GGCACCCAAGGCTT CCAAGGCTTGAAGGA(rs1347696_C8_ (rs1347696_C8_4_ GAAGGATGAAAGA TGAAAGAATGAAAGA 4_F; SEQF; SEQ ID NO: 98) ATGAAAGATGGAC TGGACGGAA G AAAAT ID NO: 97) GGAA CAAAATTAG TAGGACCTTAATTCTT GACCTTAATTCTTT TGTTCAGTTCAG (SEQ GTTCAGTTCAGID NO: 42) (SEQ ID NO: 41) rs508485 11 TTGGGGTAAATTTT TTGGGGTAAATTTTCTTGGGGTA GGGGTGGGAAT CATTGTCATATGTG ATTGTCATATGTGGA AATTTTCAT TAGACTCTGGAATTTAAATATAC ATTTAAATATACCAT TGTCA (rs508485_C11_1_ CATCATCTACAAAGCATCTACAAAGAATT (rs508485_C11_ R; SEQ ID NO100) AATTCCACAGAGTTCCACAGAGTTAAATA 1_F; SEQ AAATATCTTAAGTT TCTTAAGTTAAACAC ID NO: 99)AAACACTTAAAATA TTAAAATAAGTGTTT AGTGTTTGCGTGAT GCGTGATATTTTGAT ATTTTGATGAC AGA GA T AGATAAACAGAG TAAACAGAGTCTAA TCTAATTCCCACCCC TTCCCACCCC (SEQ(SEQ ID NO: 44) ID NO: 43) rs9788670 15 TGCAATTCAAATCA TGCAATTCAAATCAGTGCAATTCA GCAACATCGAG GGAAGTATGACCA GAAGTATGACCAAAA AATCAGGA GTTTGTCAGAAAGACAGAGATC GACAGAGATCTTTTT AGTATG (rs9788670_c15_2_ TTTTTTGGATGATCTGGATGATCCCTAGC (rs9788670_ R; SEQ ID CCTAGCCTAGCAAT CTAGCAATGCCTGGCc15_2_F; SEQ NO: 102) GCCTGGCAGCCATG AGCCATGCAGGTGCA ID NO: 101)CAGGTGCAATGTCA ATGTCAACCTTAAAT ACCTTAAATAATGT AATGTATTGCAAA T T ATTGCAAAC TCAGA CAGAGCTGACAAACC GCTGACAAACCTCG TCGATGTTGC (SEQ IDATGTTGC (SEQ ID NO: 46) NO: 45) rs8137254 22 CTGTGCTCTGCGAACTGTGCTCTGCGAAT CTGTGCTCT ACCATGCTCAT TAGCTGCAGAAGTA AGCTGCAGAAGTAACGCGAATAG GGAGAATCC ACTTGGGGACCCAA TTGGGGACCCAAAAT CTG (rs8137254_c22_2_AATAAAGCAGAAT AAAGCAGAATGCTAA (rs8137254_ R; SEQ ID GCTAATGTCAAGTCTGTCAAGTCCTGAGA c22_2_F: SEQ NO: 104) CTGAGAACCAAGC ACCAAGCCCTGGGACID NO: 103) CCTGGGACTCTGGT TCTGGTGCCATTT T G GCCATTT C GGATTCGATTCTCCATGAGCA TCCATGAGCATGGT TGGT (SEQ ID NO: 48) (SEQ ID NO: 47)rs3143 19 TTTTTCCAGCCAAC TTTTTCCAGCCAACTC TTTTTCCAG CACAGCTTGAGTCAAGGCCAAAAA AAGGCCAAAAAAAAT CCAACTCA GTTTCTTGTG AAATTTCTTAATATTTCTTAATATAGTTAT AGG (rs3143_c19_2_R; AGTTATTATGCGAG TATGCGAGGGGAGGG(rs3143_c19_2_ SEQ ID NO: 106) GGGAGGGGAAGCA GAAGCAAAGGAGCA F: SEQ IDAAGGAGCACAGGT CAGGTAGTCCACAGA NO: 105) AGTCCACAGAATA ATA G GACACAAGAA AGACACAAGAAAC ACCTCAAGCTGTG CTCAAGCTGTG (SEQ ID NO: 50) (SEQ ID NO: 49)rs2182957 13 TCTTCTCGTCCCCT TCTTCTCGTCCCCTAA TCTTCTCGT TTTCTGGTTTGTAAGCAAACAACAT GCAAACAACATCCGC CCCCTAAGC GCAACAGG CCGCTTGCTTCTGTTTGCTTCTGTCTGTGT AA (rs2182957_c13_1_ CTGTGTAACCACAG AACCACAGTGAATGG(rs2182957_ R; SEQ ID TGAATGGGTGTGCA GTGTGCACGCTTG G T c13_1_F: SEQNO: 108) CGCTTG A TGGGCCT GGGCCTCTGAGCCCC ID NO: 107) CTGAGCCCCTGTTGTGTTGCACAAACCAG CACAAACCAGAAA AAA (SEQ ID NO: 52) (SEQ ID NO: 51)rs3739005 2 CACATGGGGGCATT CACATGGGGGCATTA CACATGGG ACATCGATGAGAAGAATCGCCCAG AGAATCGCCCAGGGA GGCATTAA CACAAAAACAC GGAGGAGGAGGGAGGAGGAGGGAGAAC GAAT (rs3739005_c2_2_ GAACGCGTGCTTTT GCGTGCTTTTCACATT(rs3739005_c2_ R; SEQ ID CACATTTGCATTTG TGCATTTGAATTTT T G 2_F; SEQ IDNO: 110) AATTTT C GAGTTCC AGTTCCCAGGATGTG NO: 109) CAGGATGTGTTTTTTTTTTGTGCTCATCGA GTGCTCATCGATGT TGT (SEQ ID NO: 54) (SEQ ID NO: 53)rs530022 1 GGGCTCTGAGGTGT GGGCTCTGAGGTGTG GGGCTCTG AGATATCCCTGGTGAAATAAAAAC TGAAATAAAAACAAA AGGTGTGT GAACTGTTATT AAATGTCCATGTCTTGTCCATGTCTGTCCT GAAA CC GTCCTTTTATGGCA TTTATGGCATTTTGGG (rs530022_c1_(rs530022_c1_2_R; TTTTGGGACTTTAC ACTTTACATTTCAAA 2_F; SEQ ID SEQ ID NO: 112) ATTTCAAACATTTC CATTTCAGACATGTA NO: 111) AGACATGTATCACATCACAACACGA G GGA ACACGA A GGAATA ATAACAGTTCCAGGG ACAGTTCCAGGGATATATCT (SEQ ID ATCT (SEQ ID NO: 55) NO: 56)

Example 4 Determination of Fetal Fraction by Massively ParallelSequencing of a Target Library

To determine the fraction of fetal cIDNA in a maternal sample, targetpolymorphic nucleic acid sequences each comprising a SNP were amplifiedand used for preparing a target library for sequencing in a massivelyparallel fashion.

cfDNA was extracted as described in Example 1. A target sequencinglibrary was prepared as follows. cfDNA contained in 5 μl of purifiedcIDNA was amplified in a reaction volume of 50 μl containing 7.5 μl of a1 μM primer mix (Table 1), 10 μl of NEB 5× Mastermix and 27 μl water.Thermal cycling was performed with the Gene Amp9700 (Applied Biosystems)using the following cycling conditions: incubating at 95° C. for 1minute, followed by 20-30 cycles at 95° C. for 20 seconds, 68° C. for 1minute, and 68° C. for 30s, which was followed by a final incubation at68° C. for 5 minutes. A final hold at 4° C. was added until the sampleswere removed for combining with the unamplified portion of the purifiedcIDNA sample. The amplified product was purified using the AgencourtAMPure XP PCR purification system (Part No. A63881; Beckman CoulterGenomics, Danvers, Mass.). A final hold at 4° C. was added until thesamples were removed for preparing the target library. The amplifiedproduct was analyzed with a 2100 Bioanalyzer (Agilent Technologies,Sunnyvale, Calif.), and the concentration of amplified productdetermined A sequencing library of amplified target nucleic acids wasprepared as described in Example 2, and was sequenced in a massivelyparallel fashion using sequencing-by-synthesis with reversible dyeterminators and according to the Illumina protocol (BioTechniques®Protocol Guide 2007 Published December 2006: p 29, and on the world wideweb atbiotechniques.com/default.asp?page=protocol&subsection=article_display&id=112378).Analysis and counting of tags mapped to a reference genome consisting of26 sequences (13 pairs each representing two alleles) comprising a SNPi.e. SEQ ID NO:1-26 was performed as described.

Table 3 provides the tag counts obtained from sequencing the targetlibrary, and the calculated fetal fraction derived from sequencing data.

TABLE 3 Determination of Fetal Fraction by Massively Parallel Sequencingof a Library of Polymorphic Nucleic Acids SNP TAG Fetal SNP COUNTSFraction (%) rs10773760.1|Chr.12|length = 128|allele = A 236590 1.98rs10773760.2|Chr.12|length = 128|allele = G 4680rs13182883.1|Chr.5|length = 111|allele = A 3607 4.99rs13182883.2|Chr.5|length = 111|allele = G 72347rs4530059.1|Chr.14|length = 110|allele = A 3698 1.54rs4530059.1|Chr.14|length = 110|allele = G 239801rs8078417.1|Chr.17|length = 110|allele = C 1E+06 3.66rs8078417.2|Chr.17|length = 110|allele = T 50565 Fetal Fraction (Mean ±S.D.) = 12.4 ± 6.6

The results show that polymorphic nucleic acid sequences each comprisingat least one SNP can be amplified from cfDNA derived from a maternalplasma sample to construct a library that can be sequenced in amassively parallel fashion to determine the fraction of fetal nucleicacids in the maternal sample.

Example 5 Determination of Fetal Fraction Following Enrichment of Fetaland Maternal Nucleic Acids in a cfDNA Sequencing Library Sample

To enrich the fetal and maternal cfDNA contained in a primary sequencinglibrary constructed using purified fetal and maternal cfDNA, a portionof a purified cfDNA sample was used for amplifying polymorphic targetnucleic acid sequences, and for preparing a sequencing library ofamplified polymorphic target nucleic acids, which was used to enrich thefetal and maternal nucleic acid sequences comprised in the primarylibrary.

The method corresponds to workflow 3 diagrammed in FIG. 3. A targetsequencing library was prepared from a portion of the purified cfDNA asdescribed in Example 2. A primary sequencing library was prepared usingthe remaining portion of the purified cfDNA as described in Example 2.Enrichment of the primary library for the amplified polymorphic nucleicacids comprised in the target library was obtained by diluting theprimary and the target sequencing libraries to 10 nM, and combining thetarget library with the primary library at a ratio of 1:9 to provide anenriched sequencing library. Sequencing of the enriched library andanalysis of the sequencing data was performed as described in Example 2.

Table 4 provides the number of sequence tags that mapped to the SNPgenome for the informative SNPs identified from sequencing an enrichedlibrary derived from plasma samples of pregnant women each carrying aT21, a T13, a T18 and a monosomy X fetus, respectively. Fetal fractionwas calculated as follows:

% fetal fraction allele_(x)=((ΣFetal sequence tags forallele_(x))/(ΣMaternal sequence tags for allele_(x)))×100

Table 4 also provides the number of the sequence tags mapped to thehuman reference genome. Tags mapped to the human reference genome wereused to determine the presence or absence of aneuploidy using the sameplasma sample that was utilized for determining the corresponding fetalfraction. Method for using sequence tags counts for determininganeuploidy are described in U.S. Provisional Applications 61/407,017 and61/455,849778, which are herein incorporated by reference in theirentirety.

TABLE 4 Determination of Fetal Fraction by Massively Parallel Sequencingof an Enriched Library of Polymorphic Nucleic Acids Sample ID SNP TAGFETAL FRACTION (karyotype) SNP COUNTS (%) 11409rs13182883.1|Chr.5|length = 111|allele = A 261 4.41 (47, XY + 21)rs13182883.2|Chr.5|length = 111|allele = G 5918 rs740598.1|Chr.10|length= 114|allele = A 5545 7.30 rs740598.2|Chr.10|length = 114|allele = G 405rs8078417.1|Chr.17|length = 110|allele = C 8189 6.74rs8078417.2|Chr.17|length = 110|allele = T 121470rs576261.1|Chr.19|length = 114|allele = A 58342 7.62rs576261.2|Chr.19|length = 114|allele = C 4443 Fetal Fraction (Mean ±S.D.) = 6.5 ± 1.5 Sample ID 95133 rs1109037.1|Chr.2|length = 126|allele= A 12229 2.15 (47, XX + 18) rs1109037.2|Chr.2|length = 126|allele = G263 rs13218440.1|Chr.6|length = 139|allele = A 55949 3.09rs13218440.2|Chr.6|length = 139|allele = G 1729 rs7041158.1|Chr.9|length= 117|allele = C 7281 4.12 rs7041158.2|Chr.9|length = 117|allele = T 300rs7205345.1|Chr.16|length = 116|allele = C 53999 2.14rs7205345.2|Chr.16|length = 116|allele = G 1154 Fetal Fraction (Mean ±S.D.) = 2.9 ± 0.9 Sample ID 51236 rs13218440.1|Chr.6|length = 139|allele= A 1119 1.65 (46, XY + 13) rs13218440.2|Chr.6|length = 139|allele = G67756 rs560681.1|Chr.1|length = 111|allele = A 14123 5.18rs560681.2|Chr.1|length = 111|allele = G 732 rs7205345.1|Chr.16|length =116|allele = C 18176 1.63 rs7205345.2|Chr.16|length = 116|allele = G 296rs9866013.1|Chr.3|length = 121|allele = C 117 2.33rs9866013.2|Chr.3|length = 121|allele = T 5024 Fetal Fraction (Mean ±S.D.) = 2.7 ± 1.7 Sample ID 54430 rs1109037.1|Chr.2|length = 126|allele= A 19841 1.80 (45, XO) rs1109037.2|Chr.2|length = 126|allele = G 357rs9866013.1|Chr.3|length = 121|allele = C 12931 3.81rs9866013.2|Chr.3|length = 121|allele = T 493 rs7041158.1|Chr.9|length =117|allele = C 2800 4.25 rs7041158.2|Chr.9|length = 117|allele = T 119rs740598.1|Chr.10|length = 114|allele = A 12903 4.87rs740598.2|Chr.10|length = 114|allele = G 628 rs10773760.1|Chr.12|length= 128|allele = A 46324 4.65 rsl0773760.2|Chr.12|length = 128|allele = G2154 Fetal Fraction (Mean ± S.D.) = 3.9 ± 1.2

Example 6 Determination of Fetal Fraction by Massively ParallelSequencing Enrichment of Fetal and Maternal Nucleic Acids forPolymorphic Nucleic Acids in a Purified cfDNA Sample

To enrich the fetal and maternal cfDNA contained in a purified sample ofcfDNA extracted from a maternal plasma sample, a portion of the purifiedcfDNA was used for amplifying polymorphic target nucleic acid sequenceseach comprising one SNP chosen from the panel of SNPs given in Table 5.

The method corresponds to workflow 2 diagrammed in FIG. 3. Cell-freeplasma was obtained from a maternal blood sample, and cfDNA was purifiedfrom the plasma sample as described in Example 1. The finalconcentration was determined to be 92.8 pg/μl. cfDNA contained in 5 μlof purified cIDNA was amplified in a reaction volume of 50 μl containing7.5 μl of a 1 uM primer mix (Table 1), 10 μl of NEB 5× Mastermix and 27μl water. Thermal cycling was performed with the Gene Amp9700 (AppliedBiosystems). Using the following cycling conditions: incubating at 95°C. for 1 minute, followed by 30 cycles at 95° C. for 20 seconds, 68° C.for 1 minute, and 68° C. for 30s, which was followed by a finalincubation at 68° C. for 5 minutes. A final hold at 4° C. was addeduntil the samples were removed for combining with the unamplifiedportion of the purified cIDNA sample. The amplified product was purifiedusing the Agencourt AMPure XP PCR purification system (Part No. A63881;Beckman Coulter Genomics, Danvers, Mass.), and the concentrationquantified using the Nanodrop 2000 (Thermo Scientific, Wilmington,Del.). The purified amplification product was diluted 1:10 in water and0.9 μl (371 pg) added to 40 μl of purified cfDNA sample to obtain a 10%spike. The enriched fetal and maternal cfDNA present in the purifiedcfDNA sample was used for preparing a sequencing library, and wassequenced as described in Example 2.

Table 5 provides the tag counts obtained for each of chromosomes 21, 18,13, X and Y i.e. sequence tag density, and the tag counts obtained forthe informative polymorphic sequences contained in the SNP referencegenome . i.e. SNP tag density. The data show that sequencing informationcan be obtained from sequencing a single library constructed from apurified maternal cfDNA sample that has been enriched for sequencescomprising SNPs to simultaneously determine the presence or absence ofaneuploidy and the fetal fraction. The presence or absence of aneuploidywas determined using the number of tags mapped to chromosomes asdescribed in U.S. Provisional Applications 61/407,017 and 61/455,849. Inthe example given, the data show that the fraction of fetal DNA inplasma sample AFR105 was quantifiable from the sequencing results offive informative SNPs and determined to be 3.84%. Sequence tag densitiesare provided for chromosomes 21, 13, 18, X and Y.

The example shows that the enrichment protocol provides the requisitetag counts for determining aneuploidy and fetal fraction from a singlesequencing process.

TABLE 5 Determination of Fetal Fraction by Massively ParallelSequencing: Enrichment of Fetal and Maternal Nucleic Acids forPolymorphic Nucleic Acids in a Purified cfDNA sample AneuploidyChromosome Chromosome Chromosome Chromosome Chromosome 21 18 13 X YSequence Tag Density 178763 359529 388204 572330 2219 KaryotypeUnaffected Unaffected Unaffected Unaffected Unaffected Fetal FractionSNP SNP TAG DENSITY FETAL FRACTION (%) rs10773760.1|Chr.12|length =128|allele = A 18903 2.81 rs10773760.2|Chr.12|length = 128|allele = G532 rs1109037.1|Chr.2|length = 126|allele = A 347 5.43rs1109037.2|Chr.2|length = 126|allele = G 6394 rs2567608.1|Chr.20|length= 110|allele = A 94503 1.74 rs2567608.2|Chr.20|length = 110|allele = G1649 rs7041158.1|Chr.9|length = 117|allele = C 107 5.61rs7041158.2|Chr.9|length = 117|allele = T 6 rs8078417.1|Chr.l7|length =110|allele = C 162668 3.61 rs8078417.2|Chr.l7|length = 110|allele = T5877 Fetal Fraction (Mean ± S.D.) = 3.8 ± 1.7

Example 7 Determination of Fetal Fraction by Massively ParallelSequencing Enrichment of Fetal and Maternal Nucleic Acids forPolymorphic Nucleic Acids in a Plasma Sample

To enrich the fetal and maternal cfDNA contained in an original plasmasample derived from a pregnant woman, a portion the original plasmasample was used for amplifying polymorphic target nucleic acid sequenceseach comprising one SNP chosen from the panel of SNPs given in Table 1,and a portion of the amplified product was combined with the remainingoriginal plasma sample.

The method corresponds to workflow 2 diagrammed in FIG. 3. cIDNAcontained in 15n1 of cell-free plasma was amplified in a reaction volumeof 50 μl containing 9 μl of a 1 μM mixture of primers (15 plexTable 1),1 μl of Phusion blood DNA polymerase, 25 ul of the 2× Phusion blood PCRbuffer containing deoxynucleotide triphosphates (dNTPs: dATP, dCTP, dGTPand dTTP). Thermal cycling was performed with the Gene Amp9700 (AppliedBiosystems) using the following cycling conditions: incubating at 95° C.for 3 minutes, followed by 35 cycles at 95° C. for 20 seconds, 55° C.for 30s, and 70° C. for 1 minute, which was followed by a finalincubation at 68° C. for 5 minutes. A final hold at 4° C. was addeduntil the samples were removed for combining with the unamplifiedportion of the cell-free plasma. The amplified product was diluted 1:2with water and analyzed using the Bioanalyzer. An additional 3 μl ofamplified product was diluted with 11.85 μl of water to obtain a finalconcentration of 2 ng/μl. 2.2 μl of the diluted amplified product wascombined with the remaining plasma sample. The enriched fetal andmaternal cIDNA present in the plasma sample was purified as described inExample 1, and used for preparing a sequencing library. Sequencing andanalysis of the sequencing data was performed as described in Example 2.

The results are given in Table 6. In the example given, the data showthat the fraction of fetal DNA in plasma sample SAC2517 was quantifiablefrom the sequencing results of one informative SNP and determined to be9.5%. In the example given, sample SAC2517 was shown by karyotyping tobe unaffected for aneuploidies of chromosomes 21, 13, 18, X and Y.Sequence tag densities are provided for chromosomes 21, 13, 18, X and Y.The presence or absence of aneuploidy was determined using tag counts asdescribed in U.S. Provisional Applications 61/407,017 and 61/455,849,which are herein incorporated by reference in their entirety.

The example demonstrates that enriching the mixture of fetal andmaternal cIDNA present in a plasma sample for nucleic acid sequencesthat comprise at least one informative SNP can be used to provide therequisite sequence and SNP tag counts for determining aneuploidy andfetal fraction from a single sequencing process by massively parallelsequencing a library prepared from cfDNA contained in a plasma samplethat is enriched for polymorphic nucleic acids.

TABLE 6 Determination of Fetal Fraction by Massively ParallelSequencing: Enrichment of Fetal and Maternal Nucleic Acids forPolymorphic Nucleic Acids Comprising a SNP in a Plasma Sample AneuploidyChromosome Chromosome Chromosome Chromosome Chromosome 21 18 13 X YSequence Tag Density 183851 400582 470526 714055 2449 KaryotypeUnaffected Unaffected Unaffected Unaffected Unaffected Fetal FractionSNP TAG COUNTS FETAL FRACTION (%) rs10773760.1|Chr.12|length =128|allele = A 8536 9.5 rs10773760.2|Chr.12|length = 128|allele = G89924

Example 8 Determination of Fetal Fraction by Massively ParallelSequencing of Samples Comprising Amplified Polymorphic Sequences: TandemSNPs

To determine the fraction of fetal cIDNA in a maternal sample, targetpolymorphic nucleic acid sequences each comprising a pair of tandem SNPsare amplified and used for preparing a target library for sequencing ina massively parallel fashion. Pairs of tandem SNPs can be selected fromrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427). The primers used foramplifying the target sequences comprising the tandem SNPs are designedto encompass both SNP sites. For example, the forward primer is designedto encompass the first SNP, and the reverse primer is designed toencompass the second of the tandem SNP pair i.e. each of the SNP sitesin the tandem pair is encompassed within the 36 by generated by thesequencing method. Paired-end sequencing can be used to identify allsequences encompassing the tandem SNP sites. Exemplary sets of primersthat are used to amplify the tandem SNPs disclosed herein arers7277033-rs2110153_F (SEQ ID NOS 312 & 313): TCCTGGAAACAAAAGTATT (SEQID NO:197) and rs7277033-rs2110153_R (SEQ ID NOS 312 & 313):AACCTTACAACAAAGCTAGAA (SEQ ID NO:198), set rs2822654-rs1882882_F (SEQ IDNOS 314 & 315): ACTAAGCCTTGGGGATCCAG (SEQ ID NO:199) andrs2822654-rs1882882_R (SEQ ID NOS 314 & 315): TGCTGTGGAAATACTAAAAGG (SEQID NO:200), set rs368657-rs376635_F (SEQ ID NOS 316 &317):CTCCAGAGGTAATCCTGTGA (SEQ ID NO:201) and rs368657-rs376635_R (SEQID NOS 316 & 317):TGGTGTGAGATGGTATCTAGG (SEQ ID NO:202),rs2822731-rs2822732_F (SEQ ID NOS 318 & 319):GTATAATCCATGAATCTTGTTT (SEQID NO:203) and rs2822731-rs2822732_R (SEQ ID NOS 318 &319):TTCAAATTGTATATAAGAGAGT (SEQ ID NO:204), rs1475881-rs7275487_F (SEQID NOS 320 & 321):GCAGGAAAGTTATTTTTAAT (SEQ ID NO:205) andrs1475881-rs7275487R (SEQ ID NOS 320 & 321):TGCTTGAGAAAGCTAACACTT (SEQID NO:206), rs1735976-rs2827016F (SEQ ID NOS 322 &323):CAGTGTTTGGAAATTGTCTG (SEQ ID NO:207) and rs1735976-rs2827016R (SEQID NOS 322 & 323):GGCACTGGGAGATTATTGTA (SEQ ID NO:208),rs447349-rs2824097_F (SEQ ID NOS 324 & 325):TCCTGTTGTTAAGTACACAT (SEQ IDNO:209) and rs447349-rs2824097_R (SEQ ID NOS 324 &325):GGGCCGTAATTACTTTTG (SEQ ID NO:210), rs418989-rs13047336_F (SEQ IDNOS 326 & 327):ACTCAGTAGGCACTTTGTGTC (SEQ ID NO:211) andrs418989-rs13047336R (SEQ ID NOS 326 & 327):TCTTCCACCACACCAATC (SEQ IDNO:212), rs987980-rs987981_F (SEQ ID NOS 328 & 329):TGGCTTTTCAAAGGTAAAA(SEQ ID NO:213) and rs987980-rs987981R (SEQ ID NOS 328 & 329):GCAACGTTAACATCTGAATTT (SEQ ID NO:214), rs4143392-rs4143391_F (SEQ ID NOS330 & 331): rs4143392-rs4143391 (SEQ ID NO:215) andrs4143392-rs4143391_R (SEQ ID NOS 330 & 331):ATTTTATATGTCATGATCTAAG (SEQID NO:216), rs1691324-rs13050434_F (SEQ ID NOS 332 & 333):AGAGATTACAGGTGTGAGC (SEQ ID NO:217) and rs1691324-rs13050434R (SEQ IDNOS 332 & 333): ATGATCCTCAACTGCCTCT (SEQ ID NO:218),rs11909758-rs9980111_F (SEQ ID NOS 334 & 335): TGAAACTCAAAAGAGAAAAG (SEQID NO:219) and rs11909758-rs9980111R (SEQ ID NOS 334 & 335):ACAGATTTCTACTTAAAATT (SEQ ID NO:220), rs2826842-rs232414_F (SEQ ID NOS336 & 337): TGAAACTCAAAAGAGAAAAG (SEQ ID NO:221) and rs2826842-rs232414R(SEQ ID NOS 336 & 337): ACAGATTTCTACTTAAAATT (SEQ ID NO:222),rs2826842-rs232414_F (SEQ ID NOS 336 & 337): GCAAAGGGGTACTCTATGTA (SEQID NO:223) and rs2826842-rs232414R (SEQ ID NOS 336 & 337):TATCGGGTCATCTTGTTAAA (SEQ ID NO:224), rs1980969-rs1980970_F (SEQ ID NOS338 & 339): TCTAACAAAGCTCTGTCCAAAA (SEQ ID NO:225) andrs1980969-rs1980970R (SEQ ID NOS 338 & 339): CCACACTGAATAACTGGAACA (SEQID NO:226), rs9978999-rs9979175_F (SEQ ID NOS 340 & 341):GCAAGCAAGCTCTCTACCTTC (SEQ ID NO:227) and rs9978999-rs9979175R (SEQ IDNOS 340 & 341): TGTTCTTCCAAAATTCACATGC (SEQ ID NO:228),rs1034346-rs12481852_F (SEQ ID NOS 342 & 343): ATTTCACTATTCCTTCATTTT(SEQ ID NO:229) and rs1034346-rs12481852R (SEQ ID NOS 342 & 343):TAATTGTTGCACACTAAATTAC (SEQ ID NO:230), rs4817013-rs7277036_F: (SEQ IDNOS 346 & 347) AAAAAGCCACAGAAATCAGTC (SEQ ID NO:231) andrs4817013-rs7277036R (SEQ ID NOS 346 & 347): TTCTTATATCTCACTGGGCATT (SEQID NO:232), rs9981121-rs2829696_F (SEQ ID NOS 348 & 349):GGATGGTAGAAGAGAAGAAAGG (SEQ ID NO:233) and rs9981121-rs2829696_R (SEQ IDNOS 348 & 349): GGATGGTAGAAGAGAAGAAAGG (SEQ ID NO:234),rs455921-rs2898102_F (SEQ ID NOS 350 & 351): TGCAAAGATGCAGAACCAAC (SEQID NO:235) and rs455921-rs2898102R (SEQ ID NOS 350 & 351):TTTTGTTCCTTGTCCTGGCTGA (SEQ ID NO:236), rs2898102-rs458848_F (SEQ ID NOS352 & 353): TGCAAAGATGCAGAACCAAC (SEQ ID NO:237) and rs2898102-rs458848R(SEQ ID NOS 352 & 353): GCCTCCAGCTCTATCCAAGTT (SEQ ID NO:238),rs961301-rs2830208_F (SEQ ID NOS 354 & 355): CCTTAATATCTTCCCATGTCCA (SEQID NO:239) and rs961301-rs2830208R (SEQ ID NOS 354 & 355):ATTGTTAGTGCCTCTTCTGCTT (SEQ ID NO:240), rs2174536-rs458076_F (SEQ ID NOS356 & 357): GAGAAGTGAGGTCAGCAGCT (SEQ ID NO:241) andrs2174536-rs458076_R (SEQ ID NOS 356 & 357): TTTCTAAATTTCCATTGAACAG (SEQID NO:242), rs11088023-rs11088024_F (SEQ ID NOS 358 & 359):GAAATTGGCAATCTGATTCT (SEQ ID NO:243) and rs11088023-rs11088024_R (SEQ IDNOS 358 & 359): CAACTTGTCCTTTATTGATGT (SEQ ID NO:244),rs1011734-rs1011733_F (SEQ ID NOS 360 & 361): CTATGTTGATAAAACATTGAAA(SEQ ID NO:245) and rs1011734-rs1011733_R (SEQ ID NOS 360 & 361):GCCTGTCTGGAATATAGTTT (SEQ ID NO:246), rs2831244-rs9789838_F (SEQ ID NOS362 & 363): CAGGGCATATAATCTAAGCTGT (SEQ ID NO:247) andrs2831244-rs9789838_R (SEQ ID NOS 362 & 363): CAATGACTCTGAGTTGAGCAC (SEQID NO:248), rs8132769-rs2831440_F (SEQ ID NOS 364 & 365):ACTCTCTCCCTCCCCTCT (SEQ ID NO:249) and rs8132769-rs2831440_R (SEQ ID NOS364 & 365): TATGGCCCCAAAACTATTCT (SEQ ID NO:250), rs8134080-rs2831524_F(SEQ ID NOS 366 & 367): ACAAGTACTGGGCAGATTGA (SEQ ID NO:251) andrs8134080-rs2831524_R (SEQ ID NOS 366 & 367): GCCAGGTTTAGCTTTCAAGT (SEQID NO:252), rs4817219-rs4817220_F (SEQ ID NOS 368 & 369):TTTTATATCAGGAGAAACACTG (SEQ ID NO:253) and rs4817219-rs4817220_R (SEQ IDNOS 368 & 369): CCAGAATTTTGGAGGTTTAAT (SEQ ID NO:254),rs2250911-rs2250997_F (SEQ ID NOS 370 & 371): TGTCATTCCTCCTTTATCTCCA(SEQ ID NO:255) and rs2250911-rs2250997_R (SEQ ID NOS 370 & 371):TTCTTTTGCCTCTCCCAAAG (SEQ ID NO:256), rs2831899-rs2831900_F (SEQ ID NOS372 & 373): ACCCTGGCACAGTGTTGACT (SEQ ID NO:257) andrs2831899-rs2831900_R (SEQ ID NOS 372 & 373): TGGGCCTGAGTTGAGAAGAT (SEQID NO:258), rs2831902-rs2831903_F (SEQ ID NOS 374 & 375):AATTTGTAAGTATGTGCAACG (SEQ ID NO:259) and rs2831902-rs2831903_R (SEQ IDNOS 374 & 375): TTTTTCCCATTTCCAACTCT (SEQ ID NO:260),rs11088086-rs2251447_F (SEQ ID NOS 376 & 377): AAAAGATGAGACAGGCAGGT (SEQID NO:261) and rs11088086-rs2251447_R (SEQ ID NOS 376 & 377):ACCCCTGTGAATCTCAAAAT (SEQ ID NO:262), rs2832040-rs11088088_F (SEQ ID NOS378 & 379): GCACTTGCTTCTATTGTTTGT (SEQ ID NO:263) andrs2832040-rs11088088_R (SEQ ID NOS 378 & 379): CCCTTCCTCTCTTCCATTCT (SEQID NO:264), rs2832141-rs2246777_F (SEQ ID NOS 380 & 381): AGCACTGCAGGTA(SEQ ID NO:265) and rs2832141-rs2246777_R (SEQ ID NOS 380 & 381):ACAGATACCAAAGAACTGCAA (SEQ ID NO:266), rs2832959-rs9980934_F (SEQ ID NOS382 & 383): TGGACACCTTTCAACTTAGA (SEQ ID NO:267) andrs2832959-rs9980934_R (SEQ ID NOS 382 & 383): GAACAGTAATGTTGAACTTTTT(SEQ ID NO:268), rs2833734-rs2833735_F (SEQ ID NOS 384 & 385):TCTTGCAAAAAGCTTAGCACA (SEQ ID NO:269) and rs2833734-rs2833735_R (SEQ IDNOS 384 & 385): AAAAAGATCTCAAAGGGTCCA (SEQ ID NO:270),rs933121-rs933122_F (SEQ ID NOS 386 & 387): GCTTTTGCTGAACATCAAGT (SEQ IDNO:271) and rs933121-rs933122_R (SEQ ID NOS 386 & 387):CCTTCCAGCAGCATAGTCT (SEQ ID NO:272), rs2834140-rs12626953_F (SEQ ID NOS388 & 389): AAATCCAGGATGTGCAGT (SEQ ID NO:273) andrs2834140-rs12626953_R (SEQ ID NOS 388 & 389): ATGATGAGGTCAGTGGTGT (SEQID NO:274), rs2834485-rs3453_F (SEQ ID NOS 390 & 391):CATCACAGATCATAGTAAATGG (SEQ ID NO:275) and rs2834485-rs3453_R (SEQ IDNOS 390 & 391): AATTATTATTTTGCAGGCAAT (SEQ ID NO:276),rs9974986-rs2834703_F (SEQ ID NOS 392 & 393): CATGAGGCAAACACCTTTCC (SEQID NO:277) and rs9974986-rs2834703_R (SEQ ID NOS 392 & 393):GCTGGACTCAGGATAAAGAACA (SEQ ID NO:278), rs2776266-rs2835001_F (SEQ IDNOS 394 & 395): TGGAAGCCTGAGCTGACTAA (SEQ ID NO:279) andrs2776266-rs2835001_R (SEQ ID NOS 394 & 395):CCTTCTTTTCCCCCAGAATC (SEQID NO:280), rs1984014-rs1984015_F (SEQ ID NOS 396 &397):TAGGAGAACAGAAGATCAGAG (SEQ ID NO:281) and rs1984014-rs1984015_R(SEQ ID NOS 396 & 397):AAAGACTATTGCTAAATGCTTG (SEQ ID NO:282),rs7281674-rs2835316_F (SEQ ID NOS 398 & 399): TAAGCGTAGGGCTGTGTGTG (SEQID NO:283) and rs7281674-rs2835316_R (SEQ ID NOS 398 & 399):GGACGGATAGACTCCAGAAGG (SEQ ID NO:284), rs13047304-rs13047322_F (SEQ IDNOS 400 & 401): GAATGACCTTGGCACTTTTATCA (SEQ ID NO:285) andrs13047304-rs13047322_R (SEQ ID NOS 400 & 401):AAGGATAGAGATATACAGATGAATGGA (SEQ ID NO:286), rs2835735-rs2835736_F (SEQID NOS 404 & 405): CATGCACCGCGCAAATAC (SEQ ID NO:287) andrs2835735-rs2835736_R (SEQ ID NOS 404 & 405): ATGCCTCACCCACAAACAC (SEQID NO:288), rs13047608-rs2835826_F (SEQ ID NOS 406 & 407):TCCAAGCCCTTCTCACTCAC (SEQ ID NO:289) and rs13047608-rs2835826_R (SEQ IDNOS 406 & 407): CTGGGACGGTGACATTTTCT (SEQ ID NO:290),rs2836550-rs2212596_F (SEQ ID NOS 408 & 409): CCCAGGAAGAGTGGAAAGATT (SEQID NO:291) and rs2836550-rs2212596_R (SEQ ID NOS 408 & 409):TTAGCTTGCATGTACCTGTGT (SEQ ID NO:292), rs2836660-rs2836661_F (SEQ ID NOS410 & 411): AGCTAGATGGGGTGAATTTT (SEQ ID NO:293) and _R:TGGGCTGAGGGGAGATTC (SEQ ID NO:294), rs465612-rs8131220_F (SEQ ID NOS 412& 413): ATCAAGCTAATTAATGTTATCT (SEQ ID NO:295) and rs465612-rs8131220_R(SEQ ID NOS 412 & 413): AATGAATAAGGTCCTCAGAG (SEQ ID NO:296),rs9980072-rs8130031_F (SEQ ID NOS 414 & 415):TTTAATCTGATCATTGCCCTA (SEQID NO:297) and rs9980072-rs8130031_R (SEQ ID NOS 414 & 415):AGCTGTGGGTGACCTTGA (SEQ ID NO:298), rs418359-rs2836926_F (SEQ ID NOS 416& 417): TGTCCCACCATTGTGTATTA (SEQ ID NO:299) and rs418359-rs2836926_R(SEQ ID NOS 416 & 417): TCAGACTTGAAGTCCAGGAT (SEQ ID NO:300),rs7278447-rs7278858_F (SEQ ID NOS 418 & 419): GCTTCAGGGGTGTTAGTTTT (SEQID NO:301) and rs7278447-rs7278858_R (SEQ ID NOS 418 & 419):CTTTGTGAAAAGTCGTCCAG (SEQ ID NO:302), rs385787-rs367001_F (SEQ ID NOS420 & 421):CCATCATGGAAAGCATGG (SEQ ID NO:303) and rs385787-rs367001_R(SEQ ID NOS 420 & 421): TCATCTCCATGACTGCACTA (SEQ ID NO:304),rs367001-rs386095_F (SEQ ID NOS 422 & 423): GAGATGACGGAGTAGCTCAT (SEQ IDNO:305) and rs367001-rs386095_R (SEQ ID NOS 422 & 423):CCCAGCTGCACTGTCTAC (SEQ ID NO:306), rs2837296-rs2837297_F (SEQ ID NOS424 & 425): TCTTGTTCCAATCACAGGAC (SEQ ID NO:307) andrs2837296-rs2837297_R (SEQ ID NOS 424 & 425): ATGCTGTTAGCTGAAGCTCT (SEQID NO:308), and rs2837381-rs4816672_F (SEQ ID NOS 426 & 427):TGAAAGCTCCTAAAGCAGAG (SEQ ID NO:309) and rs2837381-rs4816672_R (SEQ IDNOS 426 & 427):TTGAAGAGATGTGCTATCAT (SEQ ID NO:310). Polynucleotidesequences e.g. GC clamp sequences, can be included to ensure specifichybridization of AT-rich primers (Ghanta et al., PLOS ONE 5(10):doi10.1371/journal.pone.0013184 [2010], available on the world wide webat plosone.org). An example of a GC clamp sequence that can be includedeither 5′ of the forward primer or 3′ of the reverse primer isGCCGCCTGCAGCCCGCGCCCCCCGTGCCCCCGCCCCGCCGCCGGCCCGGGCGCC (SEQ ID NO:311).Polymorphic sequences can be used alone or in combination withunamplified cfDNA to determine either fetal fraction or the presence orabsence of aneuploidy and fetal fraction in a maternal sample asdescribed for polymorphic SNP sequences. Sample preparation andenrichment of cfDNA sequencing library, a purified cfDNA sample, and aplasma sample is performed according to the method described in Examples5, 6, and 7, respectively. All sequencing libraries are prepared asdescribed in Example 2a., and sequencing is performed as described inExample 2b. Analysis of the sequencing data for the determination offetal aneuploidy is performed as described in Example 5. Concomitant tothe analysis for determining aneuploidy, the sequencing data is analyzedto determine the fetal fraction as follows. Following the transfer ofthe image and base call files to the Unix server running the Illumina“Genome Analyzer Pipeline” software version 1.51 as described in Example3a., the 36 bp reads are aligned to a ‘tandem SNP genome’ using theBOWTIE program. The tandem SNP genome is identified as the grouping ofthe DNA sequences that encompass the alleles of the 58 tandem SNP pairsdisclosed above. Only reads that mapped uniquely to the tandem SNPgenome are used for the analysis of fetal fraction. Reads that matchperfectly to the tandem SNP genome are counted as tags and filtered. Ofthe remaining reads, only reads having one or two mismatches are countedas tags and included in the analysis. Tags mapped to each of the tandemSNP alleles are counted, and the fetal fraction is determinedessentially as described in Example 6 above but accounting for tagsmapped to the two tandem SNP alleles x and y present on each of theamplified polymorphic target nucleic acid sequences that are amplifiedto enrich the samples i.e.

% fetal fraction allele_(x+y)=((ΣFetal sequence tags forallele_(x+y))/(ΣMaternal sequence tags for allele_(x+y)))×100

Only informative tandem SNPs are used to determine the fetal fraction.

Optionally, the fraction of fetal nucleic acids in the mixture of fetaland maternal nucleic acids is calculated for each of the informativeallele (allele“) as follows:

% fetal fraction allele_(x+y)=((2×ΣFetal sequence tags forallele_(x+y))/(ΣMaternal sequence tags for allele_(x+y)))×100,

to compensate for the presence of 2 sets of tandem fetal alleles, onebeing masked by the maternal background.

The percent fetal fraction is calculated for at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, or more informative sets of tandem alleles. In one embodiment,the fetal fraction is the average fetal fraction determined for at least3 informative sets of tandem alleles.

Example 9 Determination of Fetal Fraction by Massively ParallelSequencing of Samples Comprising Amplified Polymorphic Sequences: STRs

To determine the fraction of fetal cfDNA in a maternal sample, targetpolymorphic nucleic acid sequences each comprising an STR are amplifiedand used for preparing a target library for sequencing in a massivelyparallel fashion.

Peripheral blood samples are obtained from pregnant subjects, and cfDNAis purified from the plasma fraction as described in Examples 1 and 2STRs that are amplified are chosen from the codis and non-codis STRsdisclosed in Table 7, and amplification of the polymorphic STR sequencesis obtained using the corresponding sets of primers provided. Forexample, the STRs listed in Table 7 are amplified using thecorresponding primers (SEQ ID NOs: 113-197), and the amplified productis used to generate a target sequencing library. The STR targetsequencing library is prepared as described for the preparation of theSNP target library as described in Example 8. STRs CSF1PO, D13S317,D16S539, D18S51, D21S11, D2S1338D7S820, and FGA have been analyzedpreviously for determining fetal fraction, and are disclosed in U.S.Provisional applications 61/296,358 and 61/360,837.

TABLE 7 CODIS and NON-CODIS miniSTRs STR Locus (Marker ChromosomeSize Range GenBank Name) Location (bp) AccessionPrimer Sequences (Forward/Reverse) Codis miniSTR loci* CSF1P0 5q33.1 89-129 X14720 ACAGTAACTGCCTTCATAGATAG (CSF1PO_F; SEQ ID NO: 113)GTGTCAGACCCTGTTCTAAGTA (CSF1PO_R; SEQ ID NO: 114) FGA 4q31.3 125-281M64982 AAATAAAATTAGGCATATTTACAAGC (FGA_F; SEQ ID NO: 115)GCTGAGTGATTTGTCTGTAATTG(FGA_R; SEQ ID NO: 116) TH01 11p15.5  51-98D00269 CCTGTTCCTCCCTTATTTCCC(TH01_F; SEQ ID NO: 117)GGGAACACAGACTCCATGGTG(TH01_R; SEQ ID NO: 118) TPOX 2p25.3 65-101 M68651CTTAGGGAACCCTCACTGAATG(TPOX_F; SEQ ID NO: 119)GTCCTTGTCAGCGTTTATTTGC(TPOX_R; SEQ ID NO: 120) vWA 12p13.31  88-148M25858 AATAATCAGTATGTGACTTGGATTGA (vWA_F; SEQ ID NO: 121)ATAGGATGGATGGATAGATGGA(vWA_R; SEQ ID NO: 122) D3S1358 3p21.31  72-120NT_005997 CAGAGCAAGACCCTGTCTCAT(D3S1358_ F; SEQ ID NO: 123)TCAACAGAGGCTTGCATGTAT(D3S1358_ R; SEQ ID NO: 124) D5S818 5q23.2  81-117AC008512 GGGTGATTTTCCTCTTTGGT(D5S818_F; SEQ ID NO: 125)AACATTTGTATCTTTATCTGTATCCTTAT TTAT(D5S818_R; SEQ ID NO: 126) D7S8207q21.11 136-176 AC004848 GAACACTTGTCATAGTTTAGAACGAAC(D7S820_F; SEQ ID NO: 127) TCATTGACAGAATTGCACCA(D7S820_R;SEQ ID NO: 128) D8S1179 8q24.13  86-134 AF216671TTTGTATTTCATGTGTACATTCGTATC (D7S820_F; SEQ ID NO: 129)ACCTATCCTGTAGATTATTTTCACTGTG (D7S820_R; SEQ ID NO: 130) D13S317 13q31.1 88-132 AL353628 TCTGACCCATCTAACGCCTA(D13S317_F; SEQ ID NO: 131)CAGACAGAAAGATAGATAGATGATTGA (D13S317_R; SEQ ID NO: 132) D16S539 16q24.181-121 ACO24591 ATACAGACAGACAGACAGGTG(D16S539 _F; SEQ ID NO: 133)GCATGTATCTATCATCCATCTCT(D16S539_ R; SEQ ID NO: 134) D18S51 18q21.33113-193 AP001534 TGAGTGACAAATTGAGACCTT(D18S51_F; SEQ ID NO: 135)GTCTTACAATAACAGTTGCTACTATT (D18S51_R; SEQ ID NO: 136) D21S11 21q21.1153-221 AP000433 ATTCCCCAAGTGAATTGC(D21S11_F; SEQ ID NO: 137)GGTAGATAGACTGGATAGATAGACGA (D21S11_R; SEQ ID NO: 138) D2S1338 2q3590-142 AC01036 TGGAAACAGAAATGGCTTGG(D2S1338_F; SEQ ID NO: 139)GATTGCAGGAGGGAAGGAAG(D2S1338_ R; SEQ ID NO: 140) Penta D 21q22.3 94-167AP001752 GAGCAAGACACCATCTCAAGAA(Penta D_F; SEQ ID NO: 141)GAAATTTTACATTTATGTTTATGATTCTC T(Penta D_R; SEQ ID NO: 142) Penta E15q26.2 80-175 ACO27004 GGCGACTGAGCAAGACTC(Penta E_F; SEQ ID NO: 143)GGTTATTAATTGAGAAAACTCCTTACA (Penta E_R; SEQ ID NO: 144)Non-Codis miniSTR loci* D22S1045 22q12.3  82-115 AL022314 (17)ATTTTCCCCGATGATAGTAGTCT (D22S1045_F; SEQ ID NO: 145)GCGAATGTATGATTGGCAATATTTTT (D22S1045_R; SEQ ID NO: 146) D20S1082 20q13.2 73-101 AL158015 ACATGTATCCCAGAACTTAAAGTAAAC(D2051082_F; SEQ ID NO: 147) GCAGAAGGGAAAATTGAAGCTG(D20S1082_R; SEQ ID NO: 148) D20S482 20p13  85-126 AL121781 (14)CAGAGACACCGAACCAATAAGA (D20S482_F; SEQ ID NO: 149)GCCACATGAATCAATTCCTATAATAAA (D20S482_R; SEQ ID NO: 150) D18S853 18p11.31 82-104 AP005130 (11) GCACATGTACCCTAAAACTTAAAAT(D18S853_F; SEQ ID NO: 151) GTCAACCAAAACTCAACAAGTAGTAA(D185853R; SEQ ID NO: 152) D17S51301 17q25.1 114-139 AC016888 (12)AAGATGAAATTGCCATGTAAAAATA (D17S1301_F; SEQ ID NO: 153)GTGTGTATAACAAAATTCCTATGATGG (D17S1301_R; SEQ ID NO: 154) D17S974 17p13.1114-139 AC034303 (10) GCACCCAAAACTGAATGTCATA (D17S974_F; SEQ ID NO: 155)GGTGAGAGTGAGACCCTGTC (D17S974_R; SEQ ID NO: 156) D14S1434 14q32.13 70-98 AL121612 (13) TGTAATAACTCTACGACTGTCTGTCTG(D14S1434_F; SEQ ID NO: 157) GAATAGGAGGTGGATGGATGG(D14S1434_R; SEQ ID NO: 158) D12ATA63 12q23.3  76-106 AC009771 (13)GAGCGAGACCCTGTCTCAAG(D12ATA63_ F; SEQ ID NO: 159)GGAAAAGACATAGGATAGCAATTT (D12ATA63_R; SEQ ID NO: 160) D11S4463 11q25 88-116 AP002806 (14) TCTGGATTGATCTGTCTGTCC(D11S4463_ F; SEQ ID NO: 161)GAATTAAATACCATCTGAGCACTGAA (D11S4463R; SEQ ID NO: 162) D10S1435 10p15.3 82-139 AL354747 (11) TGTTATAATGCATTGAGTTTTATTCTG(D10S1435_F; SEQ ID NO: 163) GCCTGTCTCAAAAATAAAGAGATAGACA(D10S1435_R; SEQ ID NO: 164) D10S1248 10q26.3  79-123 AL391869 (13)TTAATGAATTGAACAAATGAGTGAG (D10S1248_F; SEQ ID NO: 165)GCAACTCTGGTTGTATTGTCTTCAT (D10S1248R; SEQ ID NO: 166) D9S2157 9q34.2 71-107 AL162417 (10) CAAAGCGAGACTCTGTCTCAA(D9S2157_ F; SEQ ID NO: 167)GAAAATGCTATCCTCTTTGGTATAAAT (D9S2157_R; SEQ ID NO: 168) D9S1122 9q21.2 93-125 AL161789 (12) GGGTATTTCAAGATAACTGTAGATAGG(D9S1122_F; SEQ ID NO: 169) GCTTCTGAAAGCTTCTAGTTTACC(D9S1122_R; SEQ ID NO: 170) D8S1115 8p11.21  63-96 AC090739 (9)TCCACATCCTCACCAACAC(D8S1115_F; SEQ ID NO: 171)GCCTAGGAAGGCTACTGTCAA(D8S1115_ R; SEQ ID NO: 172) D6S1017 6p21.1  81-110AL035588 (10) CCACCCGTCCATTTAGGC(D6S1017_F; SEQ ID NO: 173)GTGAAAAAGTAGATATAATGGTTGGTG (D6S1017_R; SEQ ID NO: 174) D6S474 6q21107-136 AL357514 (17) GGTTTTCCAAGAGATAGACCAATTA(D6S474_F; SEQ ID NO: 175) GTCCTCTCATAAATCCCTACTCATATC(D6S474R; SEQ ID NO: 176) D5S2500 5q11.2  85-126 AC008791 (17)CTGTTGGTACATAATAGGTAGGTAGGT (D5S2500_F; SEQ ID NO: 177)GTCGTGGGCCCCATAAATC(D5S2500_R; SEQ ID NO: 178) D4S2408 4p15.1  85-109AC110763 (9) AAGGTACATAACAGTTCAATAGAAAGC (D4S2408_F; SEQ ID NO: 179)GTGAAATGACTGAAAAATAGTAACCA (D4S2408R; SEQ ID NO: 180) D4S2364 4q22.3 67-83 AC022317 (9) CTAGGAGATCATGTGGGTATGATT(D4S2364U_F; SEQ ID NO: 181) GCAGTGAATAAATGAACGAATGGA(D4S2364_R; SEQ ID NO: 182) D3S4529 3p12.1 111-139 AC117452 (13)CCCAAAATTACTTGAGCCAAT(D3S452_F; SEQ ID NO: 183) GAGACAAAATGAAGAAACAGACAG(D3S452R; SEQ ID NO: 184) D3S3053 3g26.31  84-108 AC069259 (9)TCTTTGCTCTCATGAATAGATCAGT (D3S3053_F; SEQ ID NO: 185)GTTTGTGATAATGAACCCACTCAG (D3S3053_R; SEQ ID NO: 186) D2S1776 2q24.3127-161 AC009475 (11) TGAACACAGATGTTAAGTGTGTATATG(D2S1776_F; SEQ ID NO: 187) GTCTGAGGTGGACAGTTATGAAA(D2S1776_R; SEQ ID NO: 188) D2S441 2p14  78-110 AC079112 (12)CTGTGGCTCATCTATGAAAACTT(D2S441_ F; SEQ ID NO: 189)GAAGTGGCTGTGGTGTTATGAT (D2S441_ R; SEQ ID NO: 190) D1S1677 1q23.3 81-117 AL513307 (15) TTCTGTTGGTATAGAGCAGTGTTT(D1S1677_F; SEQ ID NO: 191) GTGACAGGAAGGACGGAATG(D1S1677_R; SEQ ID NO: 192) D1S1627 1p21.1  81-100 AC093119 (13)CATGAGGTTTGCAAATACTATCTTAAC (D1S1627_F; SEQ ID NO: 193)GTTTTAATTTTCTCCAAATCTCCA (D1S1627_R; SEQ ID NO: 194) D1GATA113 1p36.23 81-105 Z97987 (11) TCTTAGCCTAGATAGATACTTGCTTCC(D1GATA113_F; SEQ ID NO: 195) GTCAACCTTTGAGGCTATAGGAA(D1GATA113_R; SEQ ID NO: 196) *(Butler et al., J Forensic Sci 5:1054-1064; Hill et al., Poster #44-17th Internation Symposium on HumanIdentification-2006)

Sequencing of the library enriched for polymorphic STR sequences isperformed using a NGS technology e.g. sequencing by synthesis. Sequencereads of lengths that encompass the STRs e.g. miniSTRs of at least 100bp, to a reference STR genome consisting of the polymorphic sequenceswhich were amplified in the sample. Informative STR alleles areidentified by differences in the length of the repeats, and the numberof STR sequence tags are counted, and used to determine the fetalfraction. Optionally, amplification of the polymorphic STR sequences isperformed to enrich a plasma sample, a purified cfDNA sample or a cfDNAsequencing library sample, as described in Examples 5, 6, and 7,respectively.

Example 10 Determination of Fetal Fraction by Capillary Electrophoresisof Polymorphic Sequences Comprising STRs

To determine fetal fraction in maternal samples comprising fetal andmaternal cfDNA, peripheral blood samples were collected from volunteerpregnant women carrying either male or female fetuses. Peripheral bloodsamples were obtained and processed to provide purified cfDNA asdescribed in Example 1

Ten microliters of cfDNA samples were analyzed using the AmpF1STR®MiniFiler™ PCR amplification kit (Applied Biosystems, Foster City,Calif.) according to the manufacturer's instructions. Briefly, cfDNAcontained in 10 μl was amplified in a reaction volume of 25 μlcontaining 50_, fluorescently labeled primers (AmpF/STRO MiniFiler™Primer Set), and the AmpF/STR® MiniFiler™ Master Mix, which includesAmpliTaq Gold® DNA polymerase and associated buffer, salt (1.5 mMMgC12), and 200 μM deoxynucleotide triphosphates (dNTPs: dATP, dCTP,dGTP and dTTP). The fluorescently labeled primers are forward primersthat are labeled with 6FAM™, VIC™, NED™, and PET™ dyes. Thermal cyclingwas performed with the Gene Amp9700 (Applied Biosystems) using thefollowing cycling conditions: incubating at 95° C. for 10 minutes,followed by 30 cycles at 94° C. for 20 seconds, 59° C. for 2 minute, and72° C. for 1 minute, which was followed by a final incubation at 60° C.for 45 minutes. A final hold at 4° C. was added until the samples wereremoved for analysis. The amplified product was prepared by diluting 1ul of amplified product in 8.7 ul Hi-Di™ formamide (Applied Biosystems)and 0.3 μl GeneScan™-500 LIZ_internal size standard (AppliedBiosystems), and analyzed with an ABI PRISM3130x1 Genetic Analyzer(Applied Biosystems) using Data Collection HID_G5_POP4 (AppliedBiosystems), and a 36-cm capillary array. All genotyping was performedwith GeneMapper_ID v3.2 software (Applied Biosystems) using manufacturerprovided allelic ladders and bins and panels.

All genotyping measurement were performed on the Applied Biosystems3130x/Genetic Analyzer, using a ±0.5-nt “window” around the sizeobtained for each allele to allow for detection and correct assignmentof alleles. Any sample allele whose size was outside the ±0.5-nt windowwas determined to be OL i.e. “Off Ladder”. OL alleles are alleles of asize that is not represented in the AmpF/STR® MiniFiler™ Allelic Ladderor an allele that does not correspond to an allelic ladder, but whosesize is just outside a window because of measurement error. The minimumpeak height threshold of >50 RFU was set based on validation experimentsperformed to avoid typing when stochastic effects are likely tointerfere with accurate interpretation of mixtures. The calculation offetal fraction is based on averaging all informative markers.Informative markers are identified by the presence of peaks on theelectropherogram that fall within the parameters of preset bins for theSTRs that are analyzed.

Calculations of fetal fraction were performed using the average peakheight for major and minor alleles at every STR locus determined fromtriplicate injections. The rules applied to the calculation are:

1. off-ladder allele (OL) data for alleles are not included in thecalculation; and

2. only peak heights derived from >50 RFU (relative fluorescence units)are included in the calculation

3. if only one bin is present the marker is deemed non-informative; and

4. if a second bin is called but the peaks of the first and second binsare within 50-70% of their relative fluorescence units (RFU) in peakheight, the minority fraction is not measured and the marker is deemednot informative.

The fraction of the minor allele for any given informative marker iscalculated by dividing the peak height of the minor component by the sumof the peak height for the major component, and expressed as a percentwas first calculated for each informative locus as

fetal fraction=(Σpeak height of minor allele/Σ peak height of majorallele(s))×100,

The fetal fraction for a sample comprising two or more informative STRs,would be calculated as the average of the fetal fractions calculated forthe two or more informative markers.

Table 8 provides the data obtained from analyzing cIDNA of a subjectpregnant with a male fetus.

TABLE 8 Fetal Fraction Determined in cfDNA of a Pregnant Subject byAnalysis of STRs Allele 1 Allele 2 Allele 3 Fetal Fetal Fraction STRAllele 1 Allele 2 Allele 3 Height Height Height Fraction (Mean/STR) AMELX Y 3599 106 2.9 AMEL X Y 3602 110 3.1 AMEL X Y 3652 109 3.0 3.0 CSF1PO11 12 2870 2730 CSF1PO 11 12 2924 2762 CSF1PO 11 12 2953 2786 D13S317 1112 2621 2588 D13S317 11 12 2680 2619 D13S317 11 12 2717 2659 D16S539 911 1056 1416 D16S539 9 11 1038 1394 D16S539 9 11 1072 1437 D18S51 13 152026 1555 D18S51 13 15 2006 1557 D18S51 13 15 2050 1578 D21S11 28 31.22450 61 2.5 D21S11 28 31.2 2472 62 2.5 D21S11 28 31.2 2508 67 2.7 2.6D2S1338 20 23 3417 3017 D2S1338 20 23 3407 3020 D2S1338 20 23 3493 3055D7S820 9 12 13 2373 178 1123 5.1 D7S820 9 12 13 2411 181 1140 5.1 D7S8209 12 13 2441 182 1156 5.1 5.1 FGA 17.2 22 25 68 1140 896 3.3 FGA 17.2 2225 68 1144 909 3.1 FGA 17.2 22 25 68 1151 925 3.3 3.2 Fetal Fraction =3.5

The results show that cIDNA can be used for determining the presence orabsence of fetal DNA as indicated by the detection of a minor componentat one or more STR alleles, for determining the percent fetal fraction,and for determining fetal gender as indicated by the presence or absenceof the Amelogenin allele.

Example 11 Preamplification of cfDNA for Determining Fetal Fraction byCapillary Electrophoresis of Polymorphic Sequences Comprising STRs

To improve the sensitivity of the STR assay in detecting and quantifyingthe STR alleles in the minor contributor of the cfDNA sample, the numberof starting genomes in the artificial samples was increased by amodified whole genome amplification strategy.

Peripheral blood samples were collected and processed as described inExample 2. Cell-free DNA was extracted from 1 ml cell-free plasma usingthe Roche MagNA Pure Compact Nucleic Acid Isolation Kit I—Large Volume(Roche Applied Science, IN) using the MagNA Pure Compact Instrument, andeluted in 50 μl of elution buffer. Ten microliters of the extractedcIDNA were used to quantify the cIDNA, and the remainder was stored (seestorage instructions WI0035 Clinical Sample Storage). The concentrationof the plasma extracted cfDNA was determined by fluorescence-basedquantitation with UV absorbance measurements using the Qubit™Quantitation Platform (Invitrogen).

The concentration of cfDNA quantified in plasma samples prepared usingthe MagnaPure Nucleic Acid Isolation Kit I from 16 pregnant subjects wasdetermined to range between 20 and 100 pg/μl. As the fetal component ofplasma cfDNA is known to contribute 3-10% of the total plasma cfDNA,artificial plasma samples were created by spiking aliquots of cfDNAderived from plasma of female volunteer subjects with cfDNA extractedfrom plasma of male volunteer subjects to mimic the ratios of fetal tomaternal cIDNA found in the pregnant subjects. Artificial samples werecreated to contain 200-1000 pg of extracted female cIDNA that was spikedwith 45-150 pg of extracted male cIDNA in a total volume of 10 μl. Eachartificial sample was spiked to contain 3%, 5% and 10% male cIDNA.

Artificial samples having concentrations of total cfDNA of less thanapproximately 50 pg/μl, were pre-amplified using the modified improvedprimer extension amplification PCR (mIPEP) amplification according tothe method of Hanson and Ballantyne, (Hanson and Ballantyne, AnalyticalBiochem 346:246-257 [2005]) as follows. Ten microliters of spiked plasmacIDNA were amplified in a 25 μl reaction volume containing 1 mM dNTPs,2.5 mM MgCl₂ (Applied Biosystems), 1× Expand High Fidelity Buffer(No.3), 10.5 U Expand High Fidelity Enzyme Mix (Roche Diagnostics), and40 μM PEP primer (5′-NNNNNNNNNNNNNNN-3′, Qiagen). The amplification wasperformed in a GeneAmp PCR System 9700 Thermocycler under the followingconditions: (1) 20 and 30 cycles of 94° C. for 1 minute, 37° C. for 2minutes, and 0.1° C./s ramp to 55° C. for 4 minutes. The amplificationproduct was purified using a Qiagen column. The concentration of theamplification product was determined using the Qubit™ QuantitationPlatform as described above. STR analysis was performed as described inExample 9 above, except that only peak heights >100 RFU were included inthe calculations.

The results are shown in Tables 9, 10 and 11. The results provided inTable 9 show that the cIDNA contained in 10 μl cfDNA of artificialsamples ART23 and ART24 having a starting concentration of cIDNA of 46.2and 50.2 pg/μl, respectively, was amplified by approximately 5 and 10fold following 20 and 30 cycles of PCR amplification, respectively.

These data indicate that a pre-amplification of cIDNA using the mIPEPmethod provided enhanced levels of total cIDNA rendering the level ofthe minor component more amenable to the STR analysis.

TABLE 9 Preamplification with mIPEP cfDNA with cfDNA with cfDNA withoutmIPEP: 20 PCR mIPEP: 30 PCR SAMPLE mIPEP (pg/μl) cycles (pg/50 μl)cycles (pg/50 μl) ART23 46.2 2265 4125 ART24 50.2 2085 3875

Table 10 shows triplicate measurements profiling 9 loci of the cfDNA ofspiked samples ART23 and ART24 following the mIPEP procedure with 20 and30 cycles of amplification, as described above.

The data in Table 11 indicate that pre-amplification of cfDNA enablesthe detection and quantification of the minor component at most locitested in artificially mixed samples having a starting cfDNAconcentration that would otherwise not permit an accurate analysis ofthe minor STR alleles.

TABLE 10 mIPEP Preamplification and Detection of Minor Component ART23(453pg) ART23 (825pg) ART23 (462pg) ART24 (417pg) ART24 (775pg) ART24(502pg) mIPEP mIPEP Extracted mIPEP mIPEP Extracted amplified amplifiedunamplified amplified amplified unamplified STR 20 cycles 20 cyclescfDNA 30 cycles 30 cycles cfDNA Locus Allele Allele Height Allele HeightAllele Height Allele Allele Height Allele Height Allele Height AMEL X/Y291/95  397/170 535/832 X/Y 695/359 1878/1148 1564/1959 AMEL X/Y 425/147428/188  675/1048 X/Y 1216/619  1551/954  1573/1943 AMEL X/Y 267/94 455/203  664/1043 X/Y 718/363 1479/924  1621/2024 CSF1PO 10/11 800/979 725/1009 1429/1325 11/12 2029/1317 4159/2317 2990/3083 CSF1PO 10/111147/1432  789/1102 1779/1650 11/12 3449/2223 3460/113/1890 2996/3118CSF1PO 10/11 729/906  831/1162 1783/1657 11/12 2006/1309 3362/18403072/3183 D13S317 12 743 515 1229 11 955 1490 3634 D13S317 12 1079  5631534 11 1631  1198 3631 D13S317 12 668 583 1520 11 968 1170 3795 D16S539 9/10 239/140 370/466 835/676 10/11 513/512 1173/1472 1678/973  D16S539 9/10 347/203 64*(OL)/ 1046/864  10/11 859/870  973/1212 1730/999 391/489 D16S539  9/10 227/134 441/515 1055/860  10/11 530/513  960/11831784/1044 D18S51 14/15 359/464 363/220 785/541 12/18 1044/576  1840/786 2559/1507 512/645 391/226 999/672 12/18 1769/994  1511/643  2565/1469313/402 409/245 994/685 12/18 1033/567  1496/631  2643/1523 D21S11 29/32103/104 114/173 605/413 31.2 381  661 3276 149/153 130/182 759/523 31.2650  536 3028 85/86 131/196 760/525 31.2 380  520 3282 D2S1338 18/20572/383 428/363 1116/1013 19/20 1066/433  2315/1243 2962/2968 827/553454/386 1428/1279 19/20 1821/757  1901/101  2942/2942 530/351 482/4081431/1275 19/20 1063/444  1859/1012 3072/3067 D7S820 11/12 262/167149/270 557/627 11/12 256/138 520/322 1550/1548 62/366/231 162/292699/775 11/12 448/236 419/258 1484/1466 224/146 169/307 689/779 11/12253/141 406/250 1579/1573 FGA 21/23 263/146 181/88  596/365 22/24228/244 375/429 1272/1064 384/215 191/92  762/450 22/24 409/425 303/3451221/1023 230/136 202/102 749/456 22/24 232/250 297/348 1298/1087 *“OL”means “Off Ladder measurement”

TABLE 11 Fetal Fraction Determined in a Sample FollowingPreamplification Using mIPEP Percent minor Percent minor STR Allele 1/Allele 2/ Allele 3/ Allele 4/ fraction/STR - fraction/STR - markerHeight Height Height Height minor >100 RFU minor <100 RFU Amelogenin X/2799  Y/207 Amelogenin  X/2751  Y/198 Amelogenin  X/3109  Y/232 X/2886  Y/212 7 CSF1PO  10/2377  11/1869 12/508 CSF1PO  10/2299 11/1814 12/498 CSF1PO  10/2616 11/206 12/562  10/2431  11/1917 12/52312 D13S317  10/1232  11/1600 13/186 D13S317  10/1208  11/1548 13/182D13S317  10/1386  11/1758 13/212  10/1275  11/1635 13/193 12 D16S53911/757 12/933 D16S539 11/729 12/885 D16S539 11/836  12/1031 11/77412/950 12 D18S51 OL/80   14/3137 15/371 D18S51 11/73   14/3082 15/362D18S51 OL/83   14/3488 15/413 OL  14/3236 15/382 D21S11 29/953 30/941D21S11 29/921 30/908 D21S11  29/1046  30/1045 29/973 30/965 D2S133817/461 18/366  20/2280 24/1760 D2S1338 17/460 18/360  20/2240 24/1712D2S1338 17/508 18/409  20/2563 24/1971 17/476 18/378  20/2361 24/1814 20D7S820  8/1409 9/60  12/1059 D7S820  8/1380 9/60  12/1036 D7S820  8/15619/69  12/1166  8/1450 9/63  12/1087 2 FGA 19/825 21/850 25/279 FGA19/807 21/841 25/265 FGA 19/913 21/958 25/306 19/848 21/883 25/283 16 %fetal fraction >100RFU for minor allele →12 12 % fetal fractionincluding <100RFU for minor allele →11 11

Example 12 Correlation of Fetal Fraction Determined by Analysis of Fetaland Maternal SNPs and STRs

To verify that the calculated fetal fraction i.e. fraction of minornucleic acid component, determined using the SNP and STR assay asdescribed in the preceding Examples provided an accurate measurement ofthe fetal fraction, the percent fetal fraction of cfDNA in the plasmafrom the same pregnant subjects was compared.

Peripheral blood samples were obtained from 48 volunteer subjects, 24 ofthe subjects were pregnant with male fetuses, and 24 were pregnant withfemale fetuses. cfDNA was prepared as described in Example 1, Fetalfraction using SNPs was determined by massively parallel sequencing bysynthesis as described in Example 5, and fetal fraction using STRs wasdetermined using capillary electrophoresis as described in Example 10

The results shown in FIG. 6 indicate that a positive correlation existsbetween the fraction determined using the STR assay and the fractiondetermined using the SNP sequencing. These data further validate the useof polymorphic sequences comprising STRs or SNPs for determining thefraction of fetal cfDNA in a plasma sample.

Example 13 Use of Fetal Fraction to Set Thresholds and Estimate MinimumSample Size in Aneuploidy Detection

Counts of sequence matches to different chromosomes are manipulated togenerate a score which will vary with chromosomal copy number that canbe interpreted to identify chromosomal amplification or deletion. Forexample, such a score could be generated by comparing the relativeamount of a sequence tags on a chromosome undergoing copy number changesto a chromosome known to be a euploid. Examples of scores that can beused to identify amplification or deletion include but are not limitedto: counts for the chromosome of interest divided by counts of anotherchromosome from the same experimental run, the counts for the chromosomeof interest divided by the total number of counts from the experimentalrun, comparison of counts from the sample of interest versus a separatecontrol sample. Without loss of generality, it can be assumed thatscores will increase as copy number increases. Knowledge of fetalfraction can be used to set “cutoff” thresholds to call “aneuploidy”,“normal”, or “marginal” (uncertain) states. Then, calculations areperformed to estimate the minimum number of sequences required toachieve adequate sensitivity (i.e. probability of correctly identifyingan aneuploidy state).

FIG. 7 is a plot of two different populations of scores. The x-axis isscore and the y-axis is frequency. Scores on samples of chromosomeswithout aneuploidy can have a distribution shown in FIG. 7A. FIG. 7Billustrates a hypothetical distribution of a population of scores onsamples with an amplified chromosome. Without loss of generality, thegraphs and equations show the case of a univariate score where theaneuploidy condition represents an amplification of copy number.Multivariate cases and/or reduction/deletion abnormalities are simpleextensions or rearrangements of the given descriptions and are intend tofall within the scope of this art.

The amount of “overlap” between the populations can determine how wellnormal and aneuploidy cases can be discriminated. In general, increasingfetal fraction, ff, increases discrimination power by separating the twopopulation centers (by moving “C2,” the “Center of Aneuploidy Scores”,and increasing “d,” causing the populations to overlap less.Furthermore, an increase in the absolute value of the magnitude, m, (forexample having four copies of the chromosome instead of a trisomy) ofthe amplification will also increase separation of population centersleading to higher power (i.e. higher probability of correctlyidentifying aneuploidy states).

Increasing the number of sequences generated, N, reduces standarddeviations “sdevA” and/or “sdevB,” the spread of the two populations ofscores, which also causes the populations to overlap less.

Setting Thresholds and Estimating Sample Size

The following procedure can be used to set “c”, the critical value forcalling “aneuploidy”, “normal”, or “marginal” (uncertain) states.Without loss of generality, one sided statistical tests are used below.

First, an acceptable false positive rate, FP (sometimes also called“type I error” or “specificity”), is decided, which is the probabilityof a false positive or falsely calling aneuploidy. For example, FP canbe at least, or about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007,0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or0.1.

Second, the value of “c” can be determined by solving the equation:FP=integral from c to infinity of (f1(x)dx).

$\begin{matrix}{{F\; P} = {\int_{c}^{\infty}{f\; 1(x){x}}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

Once a critical value, c, has been determined, the minimum numbersequences required to achieve a certain TP=True positive rate can beestimated. The true positive rate can be, for example, about 0.5, 0.6,0.7, 0.8, or 0.9. In one embodiment, the true positive rate can be 0.8.In other words, N is the minimum number of sequences required toidentify aneuploidy 100*TP percent of the time. N=minimum number suchthat TP=integral from c to infinity of f2(x,ff)dx>0.8. N is determinedby solving

$\begin{matrix}{\min_{N}\mspace{14mu} {s.t.\left\{ {{T\; B} \geq {\int_{c}^{\infty}{f\; 2\left( {x,N} \right){x}}}} \right\}}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

In classical statistical tests f1 and f2 are often F, non-central Fdistributions (a special case of t and non-central t distributions)although that is not a necessary condition for this application.

Setting “Levels” of Thresholds to Give More Control of Errors

Thresholds can also be set in stages using the above methods. Forexample, a threshold can be set for high confidence calling of“aneuploidy”, say ca, using FP 0.001 and a “marginal” threshold, say cb,using FP 0.05. In this case if Score, S:

-   -   (S>ca) then call “Trisomy”    -   (cb>S<=ca) then call “Marginal”    -   (S<cb) then call “Normal”        Some Trivial Generalizations Falling Within Scope of this Art

Different values for thresholds such as TP, FP, etc can be used.Procedures can be run in any order. For example, one can start with Nand solve for c, etc. Distributions can depend on ff so that f1(x,N,ff),f2(x,N,ff), and/or other variables. The above integral equations can besolved by reference to tables or by iterative computer methods. Anon-centrality parameter can be estimated and power can be read fromstandard statistical tables. Statistical power and sample sizes may bederived from calculation or estimation of expected mean squares. Closedform theoretical distributions such as f, t, non-central t, normal, etc.or estimates (kernel or other) can be used to model the distributionsf1, f2. Empirical threshold setting and parameter selection usingReceiver Operator Characteristic Curves (ROC) can be used and collatedwith fetal fraction. Various estimates of distribution spread (variance,mean absolute deviation, inter quartile range, etc.) may be used.Various estimates of distribution center (mean, median, etc.) can beused. Two sided as opposed to one sided statistical tests can be used.The simple hypothesis test can be reformulated as linear or non-linearregression. Combinatorial methods, simulation (e.g., Monte Carlo),maximization (e.g., expectation maximization), iterative, or othermethods can be used independently or in conjunction with the above toestablish statistical power or thresholds.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for determining the fraction of fetal nucleic acids in amaternal sample comprising a mixture of fetal and maternal genomic DNA,said method comprising: (a) amplifying a plurality of polymorphic targetnucleic acids in said mixture; (b) performing massively parallelsequencing of at least a portion of the amplified product obtained in(a), wherein said sequencing comprises providing a plurality of sequencetags); and (c) based on said sequencing, determining said fraction. 2.The method of claim 1, wherein each of said plurality of polymorphictarget nucleic acids comprises at least one single nucleotidepolymorphism (SNP).
 3. The method of claim 1, wherein each of saidplurality of polymorphic target nucleic acid comprises at least oneshort tandem repeat (STR).
 4. The method of claim 1, wherein saidmassively parallel sequencing is sequencing-by-synthesis with reversibledye terminators.
 5. The method of claim 1, wherein said massivelyparallel sequencing is sequencing-by-ligation.
 6. The method of claim 1,wherein said sequencing is single molecule sequencing.
 7. The method ofclaim 1, wherein said sequencing comprises an amplification.
 8. Themethod of claim 1, wherein said maternal sample is selected from blood,plasma, serum, urine and saliva.
 9. The method of claim 1, wherein saidfetal and maternal genomic DNA is cell-free DNA (cfDNA).
 10. The methodof claim 1, wherein determining said fraction comprises determining thenumber of fetal and maternal sequence tags mapped to a reference genomecomprising said at least one polymorphic nucleic acid.
 11. The method ofclaim 2, wherein said at least one SNP is a single SNP selected fromrs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013(SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ IDNOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 &14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17 & 18),rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261(SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ IDNOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 &32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36),rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696(SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ IDNOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 &50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), andrs530022 (SEQ ID NOS 55 & 56).
 12. The method of claim 2, wherein saidat least one SNP is a tandem SNP selected from tandem SNP pairsrs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ IDNOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317);rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ IDNOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323);rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ IDNOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329);rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ IDNOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335);rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ IDNOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341);rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ IDNOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347);rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ IDNOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353);rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ IDNOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359);rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ IDNOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365);rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ IDNOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371);rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ IDNOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377);rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ IDNOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383);rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ IDNOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389);rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395);rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ IDNOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401);rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ IDNOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407);rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ IDNOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413);rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ IDNOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419);rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); andrs2837381-rs4816672 (SEQ ID NOS 426 & 427).
 13. The method of claim 3,wherein said at least one STR is selected from CSF1PO, FGA, TH01, vWA,D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482, D18S853,D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248,D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364,D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113. 14.A method for determining the fraction of fetal nucleic acids in amaternal plasma sample comprising a mixture of fetal and maternalgenomic DNA, said method comprising: (a) amplifying a plurality ofpolymorphic nucleic acids in said mixture of fetal and maternal nucleicacids, wherein each of said at least one polymorphic nucleic acidcomprises an STR; (b) determining the amount of fetal and maternal STRalleles at least one polymorphic nucleic acid; and (c) determining saidfraction using said amount of fetal and maternal STR alleles.
 15. Themethod of claim 14, further comprising preamplifying said mixture offetal and maternal nucleic acids.
 16. The method of claim 14, furthercomprising resolving the size of the STRs using capillaryelectrophoresis.
 17. The method of claim 14, said at least one STR isselected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16 S539, D18S51, D2S1338, Penta D, Penta E, D22S1045, D20S1082, D20 S482, D18S853, D17S1301, D175974, D14S1434, D12ATA63,D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017,D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441,D1S1677, D1S1627, and D1GATA113.
 18. The method of claim 1 or claim 14,wherein said at least one STR is the panel of STRs comprising CSF1PO,D13S317, D16 S539, D18S51, D21S11, D2S1338, D7S820 and FGA.
 19. Themethod of claim 1 or claim 14, wherein said method is a fetalgender-independent method.
 20. The method of claim 1 or claim 14,wherein said plurality of polymorphic nucleic acids are located on aplurality of different chromosomes.
 21. The method of claim 1 or claim14, wherein said plurality of polymorphic sites are located on achromosome other than chromosome 13, 18, 21, X or Y.
 22. The method ofclaim 1 or claim 14, wherein said plurality of polymorphic nucleic acidscomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or 40polymorphic sites.
 23. A composition for determining the fraction offetal cIDNA in a maternal sample, said composition comprising at leastone set of primers for amplifying at least one polymorphic nucleic acidin said mixture.
 24. The composition of claim 23, wherein said sample isa plasma sample.
 25. The composition of claim 23, wherein saidpolymorphic nucleic acid comprises an STR, a SNP and/or a tandem SNP.26. The composition of claim 23, wherein said STR is selected fromCSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D2S1338, Penta D, Penta E, D22S1045, D20 S1082, D20 S482,D18S853, D17S1301, D175974, D14S1434, D12ATA63, D11S4463, D10S1435,D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408,D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, andD1GATA113.
 27. The composition of claim 23, wherein said SNP is selectedfrom rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4),rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440(SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ IDNOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs4530059 (SEQ ID NOS 17& 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22),rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046(SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ IDNOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35& 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40),rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670(SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 &54), and rs530022 (SEQ ID NOS 55 & 56).
 28. The composition of claim 23,wherein said tandem SNP is selected from rs7277033-rs2110153 (SEQ ID NOS312 & 313); rs2822654-rs1882882 (SEQ ID NOS 314 & 315);rs368657-rs376635 (SEQ ID NOS 316 & 317); rs2822731-rs2822732 (SEQ IDNOS 318 & 319); rs1475881-rs7275487 (SEQ ID NOS 320 & 321);rs1735976-rs2827016 (SEQ ID NOS 322 & 323); rs447340-rs2824097 (SEQ IDNOS 324 & 325); rs418989-rs13047336 (SEQ ID NOS 326 & 327);rs987980-rs987981 (SEQ ID NOS 328 & 329); rs4143392-rs4143391 (SEQ IDNOS 330 & 331); rs1691324-rs13050434 (SEQ ID NOS 332 & 333);rs11909758-rs9980111 (SEQ ID NOS 334 & 335); rs2826842-rs232414 (SEQ IDNOS 336 & 337); rs1980969-rs1980970 (SEQ ID NOS 338 & 339);rs9978999-rs9979175 (SEQ ID NOS 340 & 341); rs1034346-rs12481852 (SEQ IDNOS 342 & 343); rs7509629-rs2828358 (SEQ ID NOS 344 & 345);rs4817013-rs7277036 (SEQ ID NOS 346 & 347); rs9981121-rs2829696 (SEQ IDNOS 348 & 349); rs455921-rs2898102 (SEQ ID NOS 350 & 351);rs2898102-rs458848 (SEQ ID NOS 352 & 353); rs961301-rs2830208 (SEQ IDNOS 354 & 355); rs2174536-rs458076 (SEQ ID NOS 356 & 357);rs11088023-rs11088024 (SEQ ID NOS 358 & 359); rs1011734-rs1011733 (SEQID NOS 360 & 361); rs2831244-rs9789838 (SEQ ID NOS 362 & 363);rs8132769-rs2831440 (SEQ ID NOS 364 & 365); rs8134080-rs2831524 (SEQ IDNOS 366 & 367); rs4817219-rs4817220 (SEQ ID NOS 368 & 369);rs2250911-rs2250997 (SEQ ID NOS 370 & 371); rs2831899-rs2831900 (SEQ IDNOS 372 & 373); rs2831902-rs2831903 (SEQ ID NOS 374 & 375);rs11088086-rs2251447 (SEQ ID NOS 376 & 377); rs2832040-rs11088088 (SEQID NOS 378 & 379); rs2832141-rs2246777 (SEQ ID NOS 380 & 381);rs2832959-rs9980934 (SEQ ID NOS 382 & 383); rs2833734-rs2833735 (SEQ IDNOS 384 & 385); rs933121-rs933122 (SEQ ID NOS 386 & 387);rs2834140-rs12626953 (SEQ ID NOS 388 & 389); rs2834485-rs3453 (SEQ IDNOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS 392 & 393);rs2776266-rs2835001 (SEQ ID NOS 394 & 395); rs1984014-rs1984015 (SEQ IDNOS 396 & 397); rs7281674-rs2835316 (SEQ ID NOS 398 & 399);rs13047304-rs13047322 (SEQ ID NOS 400 & 401); rs2835545-rs4816551 (SEQID NOS 402 & 403); rs2835735-rs2835736 (SEQ ID NOS 404 & 405);rs13047608-rs2835826 (SEQ ID NOS 406 & 407); rs2836550-rs2212596 (SEQ IDNOS 408 & 409); rs2836660-rs2836661 (SEQ ID NOS 410 & 411);rs465612-rs8131220 (SEQ ID NOS 412 & 413); rs9980072-rs8130031 (SEQ IDNOS 414 & 415); rs418359-rs2836926 (SEQ ID NOS 416 & 417);rs7278447-rs7278858 (SEQ ID NOS 418 & 419); rs385787-rs367001 (SEQ IDNOS 420 & 421); rs367001-rs386095 (SEQ ID NOS 422 & 423);rs2837296-rs2837297 (SEQ ID NOS 424 & 425); and rs2837381-rs4816672 (SEQID NOS 426 & 427).
 29. The composition of claim 23, wherein saiddetermining comprises massively parallel sequencing of fetal andmaternal cfDNA.
 30. The composition of claim 23, wherein said at leastone set of primers does not amplify a sequence on the Y chromosome. 31.A kit comprising the composition of claim 23.